The Influence of the Omicron Variant on RNA Extraction and RT-qPCR Detection of SARS-CoV-2 in a Laboratory in Brazil.

The emergence of SARS-CoV-2 variants can affect their detection via RT-qPCR. The Omicron variant has a greater affinity for the upper respiratory system and causes clinical conditions similar to bronchitis, as opposed to the pneumonitis-like conditions caused by other SARS-CoV-2 variants. This characteristic increases the viscosity of clinical samples collected for diagnosis. Coinciding with the arrival of the Omicron variant, we observed a failure in control gene expression in our laboratory. In this report, we have optimized a rapid nucleic acid extraction step to restore gene expression and detect the presence of the SARS-CoV-2 virus. We reevaluated 3000 samples, compared variant types occurring in different time periods, and confirmed that the presence of the Omicron variant was responsible for changes observed in the characteristics of these clinical samples. For samples with large amounts of mucus, such as those containing the Omicron variant, a modification to the nucleic acid extraction step was sufficient to restore the quality of RT-qPCR results.

Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers-probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples.

Detection of Group B Streptococcus in Brazilian pregnant women and antimicrobial susceptibility patterns.

Group B Streptococcus (GBS) is still not routinely screened during pregnancy in Brazil, being prophylaxis and empirical treatment based on identification of risk groups. This study aimed to investigate GBS prevalence in Brazilian pregnant women by culture or polymerase chain reaction (PCR) associated to the enrichment culture, and to determine the antimicrobial susceptibility patterns of isolated bacteria, so as to support public health policies and empirical prophylaxis. After an epidemiological survey, vaginal and anorectal specimens were collected from 221 consenting laboring women. Each sample was submitted to enrichment culture and sheep blood agar was used to isolate suggestive GBS. Alternatively, specific PCR was performed from enrichment cultures. Antimicrobial susceptibility patterns were determined for isolated bacteria by agar diffusion method. No risk groups were identified. Considering the culture-based methodology, GBS was detected in 9.5% of the donors. Twenty five bacterial strains were isolated and identified. Through the culture-PCR methodology, GBS was detected in 32.6% specimens. Bacterial resistance was not detected against ampicillin, cephazolin, vancomycin and ciprofloxacin, whereas 22.7% were resistant to erythromycin and 50% were resistant to clindamycin. GBS detection may be improved by the association of PCR and enrichment culture. Considering that colony selection in agar plates may be laboring and technician-dependent, it may not reflect the real prevalence of streptococci. As in Brazil prevention strategies to reduce the GBS associated diseases have not been adopted, prospective studies are needed to anchor public health policies especially considering the regional GBS antimicrobial susceptibility patterns.

Differences in resistance profiles and virulence genes among methicillin-resistant and methicillin-susceptible Staphylococcus aureus of different lineages at a public tertiary hospital.

INTRODUCTION: Staphylococcus aureus is a major nosocomial pathogen that is associated with high virulence and the rapid development of drug resistance. METHODS: We analyzed and compared the antimicrobial resistance, virulence profiles, and molecular epidemiology of 67 S. aureus strains, including 36 methicillin-sensitive (MSSA) and 31 methicillin-resistant (MRSA) strains recovered from a public hospital located in south-eastern Brazil. RESULTS: The clones circulating in this hospital presented a great diversity, and the majority of the strains were related to clones responsible for causing worldwide epidemics: these included USA100 (New York/Japan clone), USA300, and USA600. The 31 MRSA (22 SCCmecII and 9 SCCmecIV) and 36 MSSA strains exhibited low resistance against gentamicin and trimethoprim/sulfamethoxazole. No MRSA strain showed resistance to tetracycline. Virulence gene carriage was more diverse and abundant in MSSA than in MRSA. Of the evaluated adhesion-related genes, ebpS was the most prevalent in both MSSA and MRSA strains. The genes bbp and cna showed a strong association with MSSA strains. CONCLUSIONS: Our findings reinforce the idea that MSSA and MRSA strains should be carefully monitored, owing to their high pathogenic potential.

Antimicrobial-Resistance Genetic Markers in Potentially Pathogenic Gram Positive Cocci Isolated from Brazilian Soft Cheese.

Although most Brazilian dairy products meet high technological standards, there are quality issues regarding milk production, which may reduce the final product quality. Several microbial species may contaminate milk during manufacture and handling. If antimicrobial usage remains uncontrolled in dairy cattle, the horizontal transfer of antimicrobial resistance genes in foodstuffs may be of particular concern for both food producers and dairy industry. This study focused on the evaluation of putative Gram positive cocci in Minas cheese and of antimicrobial and biocide resistance genes among the isolated bacteria. Representative samples of 7 different industrially trademarked Minas cheeses (n = 35) were processed for selective culture and isolation of Gram positive cocci. All isolated bacteria were identified by DNA sequencing of the 16S rRNA gene. Antimicrobial resistance genes were screened by PCR. Overall, 208 strains were isolated and identified as follows: Enterococcus faecalis (47.6%), Macrococcus caseolyticus (18.3%), Enterococcus faecium (11.5%), Enterococcus caseliflavus (7.7%), Staphylococcus haemolyticus (7.2%), Staphylococcus aureus (4.3%), Staphylococcus epidermidis (2.9%), and Enterococcus hirae (0.5%). The genetic markers mecA (78.0%) and smr (71.4%) were the most prevalent, but others were also detected, such as blaZ (65.2%), msrA (60.9%), msrB (46.6%), linA (54.7%), and aacA-aphD (47.6%). The occurrence of opportunist pathogenic bacteria harboring antimicrobial resistance markers in the cheese samples are of special concern, since these bacteria are not considered harmful contaminating agents according to the Brazilian sanitary regulations. However, they are potentially pathogenic bacteria and the cheese may be considered a reservoir for antimicrobial resistance genes available for horizontal transfer through the food chain, manufacturing personnel and consumers.

Perfil epidemiológico, clínico e microbiológico de infecções por bactérias do grupo eskape em pacientes internados em uma unidade de terapia intensiva de um hospital de ensino de Minas Gerais / Epidemiological, clinical and microbiological profile of eskape group bacteria infections in patients admitted to an intensive care unit of a teaching hospital in Minas Gerais / Perfil epidemiológico, clínico y microbiológico de las infecciones por bacterias del grupo eskape en pacientes ingresados en una unidad de cuidados intensivos de un hospital escuela de Minas Gerais

Objetivo: descrever o perfil epidemiológico de infecções por bactérias do grupo ESKAPE em pacientes internados em uma unidade de terapia intensiva de um hospital de ensino da cidade de Juiz de Fora - MG. Métodos: Trata-se de um estudo epidemiológico observacional, descritivo de caráter retrospectivo, com participantes que adquiriram IRAS entre 2017 e 2018, dos quais foram isoladas e identificadas linhagens bacterianas pertencentes ao grupo ESKAPE no serviço de rotina bacteriológica do laboratório de microbiologia clínica. Resultados: Considerando o fenótipo de multirresistência entre as bactérias do grupo ESKAPE avaliadas, foi observada a ocorrência de 122 (88,4%) para Acinetobacter baumanii resistente aos carbapenêmicos, 87 (60,8%) para Klebsiella pneumoniae resistente aos carbapenêmicos, 57 (53,3%) para Pseudomonas aeruginosa resistente aos carbapenêmicos, 36 (55,4%) para MRSA e 19 (48,7%) para Enterobacter sp. resistente aos carbapenêmicos. Conclusões: A análise do perfil epidemiológico, clínico e microbiológico de infecções por bactérias do grupo ESKAPE em pacientes internados em uma UTI é de majorada importância para protocolos de terapia com o correto manejo de antibioticoterapia e redução da prevalência de resistência antimicrobiana.

Objective: To describe the epidemiological profile of ESKAPE group bacteria infections in patients admitted to an intensive care unit of a teaching hospital in Juiz de Fora, Minas Gerais. Methods: This is an observational, descriptive epidemiological study of retrospective nature, with participants who acquired IRAS between 2017 and 2018, from which bacterial strains belonging to the ESKAPE group were isolated and identified in the bacteriological routine service of the clinical microbiology laboratory. Results: Considering the multidrug resistance phenotype among the ESKAPE group bacteria evaluated, the occurrence of 122 (88.4%) for carbapenem- resistant Acinetobacter baumanii, 87 (60.8%) for carbapenem-resistant Klebsiella pneumoniae, 57 (53.3%) for carbapenem-resistant Pseudomonas aeruginosa, 36 (55.4%) for MRSA, and 19 (48.7%) for carbapenem-resistant Enterobacter sp. was observed. Conclusions: The analysis of the epidemiological, clinical and microbiological profile of infections caused by bacteria of the ESKAPE group in patients in an ICU is of major importance for therapy protocols with the correct management of antibiotic therapy and reduction of the prevalence of antimicrobial resistance.

Objetivo: Describir el perfil epidemiológico de las infecciones por bacterias del grupo ESKAPE en pacientes internados en la unidad de terapia intensiva de un hospital escuela de Juiz de Fora, Minas Gerais. Método: Se trata de un estudio epidemiológico observacional, descriptivo, de carácter retrospectivo, con participantes que adquirieron IRAS entre 2017 y 2018, de los cuales se aislaron e identificaron cepas bacterianas pertenecientes al grupo ESKAPE en el servicio de rutina bacteriológica del laboratorio de microbiología clínica. Resultados: Considerando el fenotipo de resistencia a múltiples fármacos entre las bacterias del grupo ESKAPE evaluadas, se observó la ocurrencia de 122 (88,4%) para Acinetobacter baumanii resistente a carbapenemes, 87 (60,8%) para Klebsiella pneumoniae resistente a carbapenemes, 57 (53,3%) para Pseudomonas aeruginosa resistente a carbapenemes, 36 (55,4%) para MRSA y 19 (48,7%) para Enterobacter sp. resistente a carbapenemes. Conclusiones: El análisis del perfil epidemiológico, clínico y microbiológico de las infecciones causadas por bacterias del grupo ESKAPE en pacientes ingresados en una UCI es de gran importancia para los protocolos terapéuticos con el correcto manejo de la antibioticoterapia y la reducción de la prevalencia de resistencias antimicrobianas.

Antimicrobial susceptibility and vaginolysin in Gardnerella vaginalis from healthy and bacterial vaginosis diagnosed women.

INTRODUCTION: Bacterial vaginosis (BV) is a syndrome related to Gardnerella vaginalis and is characterized by an imbalance in the vaginal microbiota. This work focused on the evaluation of antimicrobial susceptibility patterns and the occurrence of the vaginolysin (vly) gene in G. vaginalis isolated from BV and non-BV patients. METHODOLOGY: The vaginal secretions were collected randomly and processed for G. vaginalis isolation. The isolates were presumptively identified by ß-hemolysis and oxidase and catalase tests. Polymerase chain reaction (PCR) was performed to confirm bacterial identity and to detect the vly gene. Antimicrobial susceptibility patterns were determined. RESULTS: Of 89 patients, G. vaginalis was isolated from 42 (37 BV and 5 non-BV), and 204 isolates were selected (179 from BV and 25 non-BV). The vly gene was detected in all G. vaginalis isolated from non-BV women and in 98.3% of the bacteria from BV patients. High resistance was observed for ampicillin (54.4%), metronidazole (59.8%), tinidazole (60.3%) and secnidazole (71.6%). CONCLUSIONS: Further studies are needed to better address the role of G. vaginalis and the vly gene in BV pathogenesis.

Potential spread of multidrug-resistant coagulase-negative staphylococci through healthcare waste.

INTRODUCTION: Healthcare waste (HCW) might potentially harbor infective viable microorganisms in sanitary landfills. We investigated the antimicrobial susceptibility patterns and the occurrence of the mecA gene in coagulase-negative Staphylococcus strains (CoNS) recovered from the leachate of the HCW in an untreated sanitary landfill. METHODOLOGY: Bacterial identification was performed by physiological and molecular approaches, and minimum inhibitory concentrations (MICs) of antimicrobial drugs were determined by the agar dilution method according to CLSI guidelines. All oxacillin-resistant bacteria were screened for the mecA gene. RESULTS: Out of 73 CoNS, seven different species were identified by 16S rDNA sequencing: Staphylococcus felis (64.4%; n = 47), Staphylococcus sciuri (26.0%; n = 19), Staphylococcus epidermidis (2.7%; n = 2), Staphylococcus warneri (2.7%; n = 2), Staphylococcus lentus (1.4%; n = 1), Staphylococcus saprophyticus (1.4%; n = 1), and Staphylococcus haemolyticus (1.4%; n = 1). Penicillin was the least effective antimicrobial (60.3% of resistance; n = 44) followed by erythromycin (39.8%; n = 29), azithromycin (28.8%; n = 21), and oxacillin (16.5%; n = 12). The most effective drug was vancomycin, for which no resistance was observed, followed by gentamicin and levofloxacin, for which only intermediate resistance was observed (22%, n = 16 and 1.4%, n = 1, respectively). Among the oxacillin-resistant strains, the mecA gene was detected in two isolates. CONCLUSIONS: Considering the high antimicrobial resistance observed, our results raise concerns about the survival of putative bacterial pathogens carrying important resistance markers in HCW and their environmental spread through untreated residues discharged in sanitary landfills.

Differences in resistance profiles and virulence genes among methicillin-resistant and methicillin-susceptible Staphylococcus aureus of different lineages at a public tertiary hospital

Abstract INTRODUCTION Staphylococcus aureus is a major nosocomial pathogen that is associated with high virulence and the rapid development of drug resistance. METHODS We analyzed and compared the antimicrobial resistance, virulence profiles, and molecular epidemiology of 67 S. aureus strains, including 36 methicillin-sensitive (MSSA) and 31 methicillin-resistant (MRSA) strains recovered from a public hospital located in south-eastern Brazil. RESULTS The clones circulating in this hospital presented a great diversity, and the majority of the strains were related to clones responsible for causing worldwide epidemics: these included USA100 (New York/Japan clone), USA300, and USA600. The 31 MRSA (22 SCCmecII and 9 SCCmecIV) and 36 MSSA strains exhibited low resistance against gentamicin and trimethoprim/sulfamethoxazole. No MRSA strain showed resistance to tetracycline. Virulence gene carriage was more diverse and abundant in MSSA than in MRSA. Of the evaluated adhesion-related genes, ebpS was the most prevalent in both MSSA and MRSA strains. The genes bbp and cna showed a strong association with MSSA strains. CONCLUSIONS Our findings reinforce the idea that MSSA and MRSA strains should be carefully monitored, owing to their high pathogenic potential.

[Occurrence of clinically relevant bacteria in health service waste in a Brazilian sanitary landfill and antimicrobial susceptibility profile]. / Ocorrência de bactérias clinicamente relevantes nos resíduos de serviços de saúde em um aterro sanitário brasileiro e perfil de susceptibilidade a antimicrobianos.

Health service waste gives rise to controversy regarding its importance for human, animal and environmental health. Occurrences of clinically relevant bacteria in piles of health service waste in a sanitary landfill and their antimicrobial susceptibility profile were evaluated. Aliquots of leachate were processed for selective isolation of Staphylococcus sp, Gram-negative rods of the Enterobacteriaceae family and non-fermenters. Bacterial resistance to all the antimicrobials tested was observed in all microbial groups, including resistance to more than one drug. The results make it possible to suggest that viable bacteria in health service waste represent risks to human and animal health. Furthermore, occurrences of multiresistant strains support the hypothesis that health service waste acts as a reservoir for resistance markers, with an environmental impact. The lack of regional legislation concerning segregation, treatment and final disposal of waste may expose different populations to risks of transmission of infectious diseases associated with multiresistant microorganisms.

Detection of Group B Streptococcus in Brazilian pregnant women and antimicrobial susceptibility patterns

Group B Streptococcus (GBS) is still not routinely screened during pregnancy in Brazil, being prophylaxis and empirical treatment based on identification of risk groups. This study aimed to investigate GBS prevalence in Brazilian pregnant women by culture or polymerase chain reaction (PCR) associated to the enrichment culture, and to determine the antimicrobial susceptibility patterns of isolated bacteria, so as to support public health policies and empirical prophylaxis. After an epidemiological survey, vaginal and anorectal specimens were collected from 221 consenting laboring women. Each sample was submitted to enrichment culture and sheep blood agar was used to isolate suggestive GBS. Alternatively, specific PCR was performed from enrichment cultures. Antimicrobial susceptibility patterns were determined for isolated bacteria by agar diffusion method. No risk groups were identified. Considering the culture-based methodology, GBS was detected in 9.5 percent of the donors. Twenty five bacterial strains were isolated and identified. Through the culture-PCR methodology, GBS was detected in 32.6 percent specimens. Bacterial resistance was not detected against ampicillin, cephazolin, vancomycin and ciprofloxacin, whereas 22.7 percent were resistant to erythromycin and 50 percent were resistant to clindamycin. GBS detection may be improved by the association of PCR and enrichment culture. Considering that colony selection in agar plates may be laboring and technician-dependent, it may not reflect the real prevalence of streptococci. As in Brazil prevention strategies to reduce the GBS associated diseases have not been adopted, prospective studies are needed to anchor public health policies especially considering the regional GBS antimicrobial susceptibility patterns.

Ocorrência de bactérias clinicamente relevantes nos resíduos de serviços de saúde em um aterro sanitário brasileiro e perfil de susceptibilidade a antimicrobianos / Occurrence of clinically relevant bacteria in health service waste in a Brazilian sanitary landfill and antimicrobial susceptibility profile

Os resíduos de serviços de saúde suscitam polêmica quanto a importância para a saúde humana, animal e ambiental. Avaliou-se a ocorrência de bactérias clinicamente relevantes na pilha de resíduos de serviços de saúde em um aterro sanitário e seu perfil de susceptibilidade aos antimicrobianos. Alíquotas de chorume foram processadas para isolamento seletivo de Staphylococcus sp, bastonetes Gram negativos da família Enterobacteriaceae e não fermentadores. Resistência bacteriana a todos os antimicrobianos testados foi observada em todos os grupos microbianos, além de resistência a mais de uma droga. Os resultados permitem sugerir que bactérias viáveis nos resíduos de serviços de saúde representam riscos à saúde humana e animal. Além disso, a ocorrência de linhagens multirresistentes sustenta a hipótese dos resíduos de serviços de saúde atuarem como reservatórios de marcadores de resistência, com impacto ambiental. A falta de legislação regional de segregação, tratamento e destino de resíduos podem expor diferentes populações a riscos de transmissão de doenças infecciosas associadas a microrganismos multirresistentes.

Health service waste gives rise to controversy regarding its importance for human, animal and environmental health. Occurrences of clinically relevant bacteria in piles of health service waste in a sanitary landfill and their antimicrobial susceptibility profile were evaluated. Aliquots of leachate were processed for selective isolation of Staphylococcus sp, Gram-negative rods of the Enterobacteriaceae family and non-fermenters. Bacterial resistance to all the antimicrobials tested was observed in all microbial groups, including resistance to more than one drug. The results make it possible to suggest that viable bacteria in health service waste represent risks to human and animal health. Furthermore, occurrences of multiresistant strains support the hypothesis that health service waste acts as a reservoir for resistance markers, with an environmental impact. The lack of regional legislation concerning segregation, treatment and final disposal of waste may expose different populations to risks of transmission of infectious diseases associated with multiresistant microorganisms