Anovaginal Colonization by Group B Streptococcus and Streptococcus anginosus among Pregnant Women in Brazil and Its Association with Clinical Features.

Streptococcus agalactiae (Group B Streptococcus; GBS) is a leading cause of neonatal invasive disease worldwide. GBS can colonize the human gastrointestinal and genitourinary tracts, and the anovaginal colonization of pregnant women is the main source for neonatal infection. Streptococcus anginosus, in turn, can colonize the human upper respiratory, gastrointestinal, and genitourinary tracts but has rarely been observed causing disease. However, in the last years, S. anginosus has been increasingly associated with human infections, mainly in the bloodstream and gastrointestinal and genitourinary tracts. Although anovaginal screening for GBS is common during pregnancy, data regarding the anovaginal colonization of pregnant women by S. anginosus are still scarce. Here, we show that during the assessment of anovaginal GBS colonization rates among pregnant women living in Rio de Janeiro, Brazil, S. anginosus was also commonly detected, and S. anginosus isolates presented a similar colony morphology and color pattern to GBS in chromogenic media. GBS was detected in 48 (12%) while S. anginosus was detected in 17 (4.3%) of the 399 anovaginal samples analyzed. The use of antibiotics during pregnancy and history of urinary tract infections and sexually transmitted infections were associated with the presence of S. anginosus. In turn, previous preterm birth was associated with the presence of GBS (p < 0.05). The correlation of GBS and S. anginosus with relevant clinical features of pregnant women in Rio de Janeiro, Brazil, highlights the need for the further investigation of these important bacteria in relation to this special population.

Changes in Group B Streptococcus Colonization among Pregnant Women before and after the Onset of the COVID-19 Pandemic in Brazil.

Group B Streptococcus (GBS) is a leading cause of neonatal infections. The genitourinary and gastrointestinal tract of pregnant women are the main source of transmission to newborns. This work investigated the prevalence and characterized GBS from pregnant women in Rio de Janeiro, Brazil, comparing the periods before (January 2019 to March 2020; 521) and during (May 2020 to March 2021; 285) the COVID-19 pandemic. GBS was detected in 10.8% of anovaginal samples. Considering scenarios before and during the pandemic, GBS colonization rate significantly decreased (13.8% vs. 5.3%; p = 0.0001). No clinical and sociodemographic aspect was associated with GBS carriage (p > 0.05). A total of 80%, 13.8% and 4.6% GBS strains were non-susceptible to tetracycline, erythromycin and clindamycin, respectively. Serotype Ia was the most frequent (47.7%), followed by V (23.1%), II (18.4%), III (7.7%) and Ib (3.1%). An increasing trend of serotypes Ib and V, as well as of antimicrobial resistance rates, and a decreasing trend of serotypes II and III, were observed after the pandemic onset, albeit not statistically significant (p > 0.05). The reduction in GBS colonization rates and alterations in GBS serotypes and resistance profiles during the pandemic were not due to changes in the sociodemographic profile of the population. Considering that control and preventive measures related to the COVID-19 pandemic onset have impacted other infectious diseases, these results shed light on the need for the continuous surveillance of GBS among pregnant women in the post-pandemic era.

Assessing in vivo and in vitro biofilm development by Streptococcus dysgalactiae subsp. dysgalactiae using a murine model of catheter-associated biofilm and human keratinocyte cell.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is an important agent of bovine mastitis. This infection causes an inflammatory reaction in udder tissue, being the most important disease-causing significant impact on the dairy industry. Therefore, it leads to an increase in dairy farming to meet commercial demands. As a result, there is a major impact on both the dairy industry and the environment including global warming. Recurrent mastitis is often attributed to the development of bacterial biofilms, which promote survival of sessile cells in hostile environments, and resistance to the immune system defense and antimicrobial therapy. Recently, we described the in vitro biofilm development on abiotic surfaces by bovine SDSD. In that work we integrated microbiology, imaging, and computational methods to evaluate the biofilm production capability of SDSD isolates on abiotic surfaces. Additionally, we reported that bovine SDSD can adhere and internalize human cells, including human epidermal keratinocyte (HEK) cells. We showed that the adherence and internalization rates of bovine SDSD isolates in HEK cells are higher than those of a SDSD DB49998-05 isolated from humans. In vivo, bovine SDSD can cause invasive infections leading to zebrafish morbidity and mortality. In the present work, we investigated for the first time the capability of bovine SDSD to develop biofilm in vivo using a murine animal model and ex-vivo on human HEK cells. Bovine SDSD isolates were selected based on their ability to form weak, moderate, or strong biofilms on glass surfaces. Our results showed that SDSD isolates displayed an increased ability to form biofilms on the surface of catheters implanted in mice when compared to in vitro biofilm formation on abiotic surface. A greater ability to form biofilm in vitro after animal passage was observed for the VSD45 isolate, but not for the other isolates tested. Besides that, in vitro scanning electron microscopy demonstrated that SDSD biofilm development was visible after 4 hours of SDSD adhesion to HEK cells. Cell viability tests showed an important reduction in the number of HEK cells after the formation of SDSD biofilms. In this study, the expression of genes encoding BrpA-like (biofilm regulatory protein), FbpA (fibronectin-binding protein A), HtrA (serine protease), and SagA (streptolysin S precursor) was higher for biofilm grown in vivo than in vitro, suggesting a potential role for these virulence determinants in the biofilm-development, host colonization, and SDSD infections. Taken together, these results demonstrate that SDSD can develop biofilms in vivo and on the surface of HEK cells causing important cellular damages. As SDSD infections are considered zoonotic diseases, our data contribute to a better understanding of the role of biofilm accumulation during SDSD colonization and pathogenesis not only in bovine mastitis, but they also shed some lights on the mechanisms of prosthesis-associated infection and cellulitis caused by SDSD in humans, as well.

Influence of Penicillin on Biofilm Formation by Streptococcus agalactiae Serotype Ia/CC23.

Streptococcus agalactiae (Group B Streptococcus , GBS) is a major agent of perinatal infections. Biofilms have been associated with GBS colonization and disease, as well as with infection persistence and recurrence. Although GBS remains susceptible to beta-lactams, it is still unknown how sessile cells respond to these antibiotics. Here, we evaluated the effect of different concentrations of penicillin (3-48 mg/L) on in vitro biofilm formation by four GBS strains belonging to serotype Ia/clonal complexes23 that were recovered from the oropharynx or urine of pregnant women and were previously characterized as strong biofilm producers. All four GBS strains were fully susceptible to penicillin (minimum inhibitory concentration = 0.023 mg/L), but penicillin was not able to fully prevent biofilm formation by these GBS strains. Biofilms formed in the presence of penicillin had reduced biomasses and thickness, but they were still classified as strong. Penicillin significantly reduced the density of live cells, but higher penicillin concentrations did not lead to improved prevention of biofilm formation. Biofilms formed in the presence of penicillin had no channels or long cocci chains observed in penicillin-free biofilms. Overall, results highlight the concerning possible impacts of biofilm formation in penicillin-based treatment and preventive strategies of GBS infections, even when the bacterial strain involved is fully antibiotic-susceptible.

Biofilm production and distribution of pilus variants among Streptococcus agalactiae isolated from human and animal sources.

Streptococcus agalactiae (group B Streptococcus, GBS) is a major pathogen in humans and animals. Pili and biofilm may be important virulence factors in this bacterial species. Here, biofilm production and the distribution of pilus variants among 134 GBS isolates from human and animal sources were evaluated. Biofilm production was significantly enhanced in 1% glucose-supplemented medium (p < 0.05). Using this medium, most GBS strains were strong biofilm producers. Biomass was mainly composed of proteins, followed by extracellular DNA, while polysaccharides represented a minor portion. All GBS strains presented at least one pilus variant. PI-2a was the most common among human GBS while PI-2b was the most common among animal isolates. Human GBS harboring PI-2b and animal GBS harboring PI-2a presented significantly reduced biofilm production (p = 0.0033). In conclusion, strong biofilm production seems to be a common characteristic in GBS, and association of the clinical source with the pilus variant may be crucial for this.

Salmonella Panama: Genetic Diversity of the Isolates Collected from Human and Non-human Sources.

INTRODUCTION: Salmonella enterica serovar Panama belongs to the D1 serogroup and is frequently associated with nontyphoidal salmonellosis in humans. This study aimed to characterize isolates collected from Northeast Brazil by phenotypic and molecular methods. METHODS: Forty four S. Panama strains were examined for antimicrobial susceptibility, virulence genes, and pulsed field gel electrophoresis (PFGE) types. RESULTS: All strains were susceptible to antibiotics (except for streptomycin), presented classical virulence factors, and could be clustered into four groups and 18 pulsotypes. CONCLUSIONS: This work calls for continuous surveillance for the emergence of antibiotic resistance and new clones in a geographical area.

Salmonella Panama: genetic diversity of the isolates collected from human and non-human sources

Abstract INTRODUCTION Salmonella enterica serovar Panama belongs to the D1 serogroup and is frequently associated with nontyphoidal salmonellosis in humans. This study aimed to characterize isolates collected from Northeast Brazil by phenotypic and molecular methods. METHODS Forty four S. Panama strains were examined for antimicrobial susceptibility, virulence genes, and pulsed field gel electrophoresis (PFGE) types. RESULTS All strains were susceptible to antibiotics (except for streptomycin), presented classical virulence factors, and could be clustered into four groups and 18 pulsotypes. CONCLUSIONS This work calls for continuous surveillance for the emergence of antibiotic resistance and new clones in a geographical area.

Meningitis caused by Salmonella enterica serotype Panama in Brazil: first case reported.

Salmonella infections usually occur as gastroenteritis that is generally self-limited. However, some serotypes of Salmonella can cause severe extra-intestinal infections, such as bacteremia and meningitis. Here, we report the first Salmonella Panama case of meningitis in 4-month-old male newborn in Brazil. The invasive strain isolated was susceptible to all antimicrobial agents tested. The genes agfA, fimA, invA, sfbA, phoP, and slyA were detected using polymerase chain reactions. These findings are relevant and physicians should be alert to the possibility of meningitis in newborns due to S. Panama, which can present a high rate of mortality or recurrence of infection.

Meningitis caused by Salmonella enterica serotype Panama in Brazil: first case reported

Abstract Salmonella infections usually occur as gastroenteritis that is generally self-limited. However, some serotypes of Salmonella can cause severe extra-intestinal infections, such as bacteremia and meningitis. Here, we report the first Salmonella Panama case of meningitis in 4-month-old male newborn in Brazil. The invasive strain isolated was susceptible to all antimicrobial agents tested. The genes agfA, fimA, invA, sfbA, phoP, and slyA were detected using polymerase chain reactions. These findings are relevant and physicians should be alert to the possibility of meningitis in newborns due to S. Panama, which can present a high rate of mortality or recurrence of infection.

Antimicrobial resistance in Neisseria gonorrhoeae: history, molecular mechanisms and epidemiological aspects of an emerging global threat

ABSTRACT Neisseria gonorrhoeae is the agent of gonorrhea, a sexually transmitted infection with an estimate from The World Health Organization of 78 million new cases in people aged 15-49 worldwide during 2012. If left untreated, complications may include pelvic inflammatory disease and infertility. Antimicrobial treatment is usually effective; however, resistance has emerged successively through various molecular mechanisms for all the regularly used therapeutic agents throughout decades. Detection of antimicrobial susceptibility is currently the most critical aspect for N. gonorrhoeae surveillance, however poorly structured health systems pose difficulties. In this review, we compiled data from worldwide reports regarding epidemiology and antimicrobial resistance in N. gonorrhoeae, and highlight the relevance of the implementation of surveillance networks to establish policies for gonorrhea treatment.

Antimicrobial resistance in Neisseria gonorrhoeae: history, molecular mechanisms and epidemiological aspects of an emerging global threat.

Neisseria gonorrhoeae is the agent of gonorrhea, a sexually transmitted infection with an estimate from The World Health Organization of 78 million new cases in people aged 15-49 worldwide during 2012. If left untreated, complications may include pelvic inflammatory disease and infertility. Antimicrobial treatment is usually effective; however, resistance has emerged successively through various molecular mechanisms for all the regularly used therapeutic agents throughout decades. Detection of antimicrobial susceptibility is currently the most critical aspect for N. gonorrhoeae surveillance, however poorly structured health systems pose difficulties. In this review, we compiled data from worldwide reports regarding epidemiology and antimicrobial resistance in N. gonorrhoeae, and highlight the relevance of the implementation of surveillance networks to establish policies for gonorrhea treatment.

Staphylococcus saprophyticus Recovered from Humans, Food, and Recreational Waters in Rio de Janeiro, Brazil.

Staphylococcus saprophyticus is an important agent of urinary tract infection (UTI) in young women, but information about this pathogen in human microbiota and in common environment is lacking. The aim of this study was to characterize S. saprophyticus isolates from genitoanal microbiota of 621 pregnant women, 10 minas cheese packs, and five beaches in Rio de Janeiro city and compare PFGE profiles of these isolates with five UTI PFGE clusters described in this city. We investigated 65 S. saprophyticus isolates from microbiota, 13 from minas cheese, and 30 from beaches and 32 UTI isolates. Antimicrobial resistance was determined by disk diffusion, MIC by agar dilution, and PCR. Erythromycin-resistance genes erm(C), msr(A), msr(B), mph(C), and lin(A) were found in 93% of isolates. Trimethoprim-sulfamethoxazole resistance correlated with dfrG or dfrA genes. Three cefoxitin-resistant isolates carried the mecA gene. All isolates obtained from cheese were susceptible to all antimicrobial agents. Six of 10 pregnant women with >1 isolate had monoclonal colonization. Isolates from pregnant women shared 100% similarity with UTI PFGE cluster types A and E obtained almost 10 years previously, suggesting temporal persistence of S. saprophyticus. Antimicrobial resistance of beach isolates reflected the profiles of human isolates. Taken together, results indicate a shared source for human and environmental isolates.

Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA) has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR). Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

Impact of agr dysfunction on virulence profiles and infections associated with a novel methicillin-resistant Staphylococcus aureus (MRSA) variant of the lineage ST1-SCCmec IV.

BACKGROUND: A novel variant of the ST1-SCCmecIV methicillin-resistant Staphylococcus aureus (MRSA) lineage, mostly associated with nosocomial bloodstream infections (BSI), has emerged in Rio de Janeiro. Bacterial biofilm has been considered a major virulence factor in central venous catheter-associated BSI. The mechanisms involved in biofilm formation/accumulation are multifactorial and complex. Studies have suggested that biofilm production was affected in vitro and vivo for agr-null mutants of S. aureus. RESULTS: The impact of naturally occurring inhibition of agr signaling on virulence profiles and infections associated with the ST1 variant was investigated. agr dysfunction was detected in a significant percentage (13%) of the isolates with concomitant increase in biofilm accumulation in vitro and in vivo, and enhanced ability to adhere to and invade airway cells. The biofilm formed by these ST1 isolates was ica-independent and proteinaceous in nature. In fact, the improved colonization properties were paralleled by an increased expression of the biofilm-associated genes fnbA, spa and sasG. The transcription of sarA, a positive regulator of agr, was two-times reduced for the agr-dysfunctional MRSA. Remarkably, the agr inhibition was genetically stable. Indeed, agr-dysfunctional isolates succeed to colonize and cause both acute and chronic infections in hospitalized patients, and also to effectively accumulate biofilm in a mouse subcutaneous catheter implant model. CONCLUSION: The ability of agr-dysfunctional isolates to cause infections in humans and to form biofilm in the animal model suggests that therapeutic approaches based on agr-inactivation strategies are unlikely to be effective in controlling human-device infections caused by ST1 isolates. The increased biofilm accumulation associated with the acquisition of multiple antimicrobial resistant traits might have influenced (at least in part) the expansion of this USA400 related clone in our hospitals.

Antibacterial activity and cytotoxicity analysis of halistanol trisulphate from marine sponge Petromica citrina.

OBJECTIVES: An aqueous extract and fraction from the marine sponge Petromica citrina have antibacterial activity. We performed a chemical and biological characterization of the antibiotic substance from P. citrina and investigated its mode of action on Staphylococcus aureus cells. METHODS: The inhibitory activity of the aqueous extract of P. citrina was determined against 14 bacteria belonging to type strains and clinical antibiotic-resistant strains. The aqueous extract was fractionated under bioassay guidance and the bioactive substance was identified by its (1)H-NMR, (13)C-NMR and mass spectra. The MIC and the MBC of this substance were determined. This substance was also subjected to cytotoxic bioassays. The mode of action on S. aureus cells was investigated by light and transmission electron microscopy analysis. RESULTS: P. citrina showed a large spectrum of activity against type strains and resistant-bacteria such as S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum and Neisseria gonorrhoeae. The aqueous extract was fractionated and halistanol trisulphate (24ε,25-dimethylcholestane-2ß,3α,6α-triol trisodium sulphate) was isolated for the first time from P. citrina. Halistanol trisulphate had a bactericidal effect on exponentially growing S. aureus cells at the MIC (512 mg/L). Cytotoxicity biossays showed moderate toxicity against cancer cell line L929 (fibrosarcoma). This substance apparently acts by damaging the cell membrane, with subsequent cell lysis. CONCLUSIONS: Halistanol trisulphate is a broad-spectrum antibiotic isolated from P. citrina with a mode of action involving disruption of the cytoplasmic membrane. It is a new candidate for research on antibacterial substances.

Characterization of coagulase-negative staphylococci isolated from hospital indoor air and a comparative analysis between airborne and inpatient isolates of Staphylococcus epidermidis.

A total of 108 coagulase-negative staphylococci (CoNS) were collected from hospital indoor air. The majority of the isolates were able to produce biofilms and displayed multiresistance profiles. The most frequent species identified were Staphylococcus epidermidis (n=27) and Staphylococcus haemolyticus (n=17). Potential virulence traits (icaAD, aap, hld, atlE and sesB) and genotypic profiles were compared for S. epidermidis isolates from indoor air (n=27) and from patients (n=26) who had been admitted to the hospital 8-34 months after air sampling. Overall, the virulence factors tested were more frequently found among S. epidermidis recovered from clinical origin than from air sources (P=0.003). Indeed, the group of patient isolates exhibited superior ability to accumulate biofilms (P<0.0001). Despite this, genotyping using PFGE revealed that identical clones of S. epidermidis could be recovered from both patient and indoor air samples. In addition, some airborne isolates displayed virulence profiles and levels of biofilm accumulation similar to those found in patient isolates. Therefore, further studies are necessary to clarify the importance of hospital indoor air as a route of transmission for CoNS isolates (mainly S. epidermidis).

Comparison of in vitro and in vivo systems to study ica-independent Staphylococcus aureus biofilms.

The ability of Staphylococcus aureus to form biofilms is considered an important factor in the pathogenesis of central venous catheter-related bacteremia and infections associated with the use of medical prostheses. Different methods have been described for assessing staphylococcal biofilms, but few comparative studies have been attempted to evaluate these techniques; especially related to ica-independent biofilm formation/accumulation. In this study we compared some in vitro and in vivo techniques to evaluate ica-independent biofilms produced by methicillin-resistant S. aureus. We observed that biofilms formed on human fibronectin-covered surfaces were about three times higher than those produced on inert polystyrene surfaces. However, despite the difference in absolute values, a linear correlation was detected between these two models. We also found that biofilms formed on polystyrene or polyurethane surfaces treated with human serum were easily detachable during washing and staining processes. The mouse model of subcutaneous foreign body showed good correlation with the in vitro techniques using either inert polystyrene or solid-phase fibronectin. Thus, our data showed that the microtiter-plate-based spectrophotometric assay is an appropriate method for preliminary biofilm investigations, mainly when a large number of isolates, mutants or systems need to be tested.