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.

Can the Enterococcus faecalis identified in the root canals of primary teeth be a cause of failure of endodontic treatment?

OBJECTIVE: This study investigated the presence of Enterococcus faecalis in primary teeth with primary root canal infections and related to the possible failure of pulpectomy outcome after 36 months. MATERIAL AND METHODS: Root canal samples were obtained from 25 out of 244 patients using the sterile paper cone method. The identification of E. faecalis was done with culture and molecular tests using species-specific 16S rRNA gene-based polymerase chain reaction (PCR). After 36 months, the pulpectomy outcome was evaluated. RESULTS: Enterococcus faecalis was found in five (20%) samples, and dental caries were the cause of primary infection in all of them. Pulpectomy outcome was evaluated only in teeth that completed the entire clinical protocol and were followed up to 36 months (n = 8). From these, 75% (n = 6) were successful and 25% (n = 2) failed. E. faecalis was present in 50% of both successful and failed cases. CONCLUSIONS: Enterococcus faecalis was not related to the failure of endodontic treatment of primary teeth.

First report of the emerging zoonotic agent Wohlfahrtiimonas chitiniclastica isolated from a retail frozen chicken in Rio de Janeiro, Brazil.

Wohlfahrtiimonas chitiniclastica is an emerging zoonotic bacterium commensally living in larvae of particular flies. It has been associated with human and animal infections but never isolated from food. In the present study, a whole chicken carcass was rinsed in buffered peptone water which was then inoculated into BHI and the growth plated onto selective medium. Species identification was performed by MALDI-TOF MS. Those bacteria identified as W. chitiniclastica were subjected to 16S rRNA sequencing for confirmation and MEGA software was used to obtain their phylogenetic position. The findings of this study raise concerns regarding the abattoir, transport and stock practices of frozen meat carcasses and should be of interest with regard to microbiology, entomology and food production.

Long-Term Co-Circulation of Host-Specialist and Host-Generalist Lineages of Group B Streptococcus in Brazilian Dairy Cattle with Heterogeneous Antimicrobial Resistance Profiles.

Group B Streptococcus (GBS) is a major cause of contagious bovine mastitis (CBM) in Brazil. The GBS population is composed of host-generalist and host-specialist lineages, which may differ in antimicrobial resistance (AMR) and zoonotic potential, and the surveillance of bovine GBS is crucial to developing effective CBM control and prevention measures. Here, we investigated bovine GBS isolates (n = 156) collected in Brazil between 1987 and 2021 using phenotypic testing and whole-genome sequencing to uncover the molecular epidemiology of bovine GBS. Clonal complex (CC) 61/67 was the predominant clade in the 20th century; however, it was replaced by CC91, with which it shares a most common recent ancestor, in the 21st century, despite the higher prevalence of AMR in CC61/67 than in CC91, and high selection pressure for AMR from indiscriminate antimicrobial use in the Brazilian dairy industry. CC103 also emerged as a dominant CC in the 21st century, and a considerable proportion of herds had two or more GBS strains, suggesting poor biosecurity and within-herd evolution due to the chronic nature of CBM problems. The majority of bovine GBS belonged to serotype Ia or III, which was strongly correlated with CCs. Ninety-three isolates were resistant to tetracycline (≥8 µg/mL; tetO = 57, tetM = 34 or both = 2) and forty-four were resistant to erythromycin (2.0 to >4 µg/mL; ermA = 1, ermB = 38, mechanism unidentified n = 5). Only three isolates were non-susceptible to penicillin (≥8.0 µg/mL), providing opportunities for improved antimicrobial stewardship through the use of narrow-spectrum antimicrobials for the treatment of dairy cattle. The common bovine GBS clades detected in this study have rarely been reported in humans, suggesting limited risk of interspecies transmission of GBS in Brazil. This study provides new data to support improvements to CBM and AMR control, bovine GBS vaccine design, and the management of public health risks posed by bovine GBS in Brazil.

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.

Nasopharyngeal carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among children from Brazil before the introduction of the 10-valent conjugate vaccine.

BACKGROUND: Streptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide. Nasopharyngeal colonization plays an important role in the development and transmission of pneumococcal diseases, and infants and young children are considered to be the main reservoir of this pathogen. The aim of this study was to evaluate the rates and characteristics associated with nasopharyngeal carriage, the distribution of serotypes and the antimicrobial resistance profiles of Streptococcus pneumoniae among children in a large metropolitan area in Brazil before the introduction of the 10-valent pneumococcal conjugate vaccine. METHODS: Between March and June 2010, nasopharyngeal swabs were collected from 242 children aged <6 years attending one day care center and the emergency room of a pediatric hospital. Pneumococcal isolates were identified by conventional methods and serotypes were determined by a sequential multiplex PCR assay and/or the Quellung reaction. The antimicrobial susceptibilities of the pneumococci were assessed by the disk diffusion method. MICs for erythromycin and penicillin were also performed. Erythromycin resistance genes were investigated by PCR. RESULTS: The overall colonization rate was 49.2% and it was considerably higher among children in the day care center. Pneumococcal carriage was more common among day care attenders and cohabitants with young siblings. The most prevalent serotypes were 6B, 19F, 6A, 14, 15C and 23F, which accounted for 61.2% of the isolates. All isolates were susceptible to clindamycin, levofloxacin, rifampicin and vancomycin. The highest rate of non-susceptibility was observed for sulphamethoxazole-trimethoprim (51.2%). Penicillin non-susceptible pneumococci (PNSP) accounted for 27.3% of the isolates (MICs of 0.12-4 µg/ml). Penicillin non-susceptibility was strongly associated with serotypes 14 and 23F. Hospital attendance and the presence of respiratory or general symptoms were frequently associated with PNSP carriage. The two erythromycin-resistant isolates (MICs of 2 and 4 µg/ml) belonged to serotype 6A, presented the M phenotype and harbored the mef(A/E) gene. CONCLUSIONS: Correlations between serotypes, settings and penicillin non-susceptibility were observed. Serotypes coverage projected for the 10-valent pneumococcal conjugate vaccine was low (45.5%), but pointed out the potential reduction of PNSP nasopharyngeal colonization by nearly 20%.

Carriage prevalence, serotype distribution, antimicrobial resistance, pspA typing and pilus islets of Streptococcus pneumoniae isolated from adults living in a Brazilian urban slum.

INTRODUCTION: For Brazilian adults, pneumococcal vaccines have been usually taken only by those who are at higher risk for development of pneumococcal diseases. Since populations from lower socioeconomic status are at high risk of acquiring pneumococcal infections, we investigated the carriage prevalence, colonization risk factors, capsular and surface protein types, and antimicrobial resistance among pneumococcal isolates recovered from adults living in a Brazilian urban slum. METHODS: Between September-December 2016, we conducted a cross-sectional study among individuals aged ≥ 18 years who attended a public primary clinic in Niterói/RJ, Brazil. Pneumococci were isolated by culture on sheep blood agar plates with and without gentamicin. Antimicrobial susceptibility was determined for all isolates. We used PCR to determine capsular types, PspA families (Fam) and pilus islets (PI). RESULTS: Of 385 adults, 32 (8.3 %) were pneumococcal carriers. Three carriers had two different pneumococci, totaling 35 isolates. After multivariate analysis, smoking, previous hospitalization, alcohol consumption and co-habitation with children aged < 6 years increased the odds of pneumococcal carriage, but antibiotic use in the previous 2 weeks was found to be a protective factor. Fourteen different serogroups/serotypes were detected and the prevalent ones were 9 N/L, 10A, 15B/C and 35F/47F (n = 3; 8.6 % each). Non-typeable (NT) isolates made up 31.4 %. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. We found eight (22.9 %) penicillin non-susceptible pneumococci (PNSP) with minimum inhibitory concentrations (MICs) of 0.38-1.5 µg/mL. The two (5.7 %) erythromycin-resistant isolates had MIC > 256 µg/mL, cMLSB phenotype and the erm(B) gene. Twelve (34.3 %) and 17 (48.6 %) isolates had PspA Fam1 and Fam2, respectively. Three (8.6 %) isolates had genes for pilitwo PI-1 and one PI-2. CONCLUSION: We detected a low frequency of pneumococcal carriage among the adult population, but a high diversity of serotypes. Frequencies of PNSP and NT isolates resistant to antimicrobial agents are concerning.

Draft Genome Sequences of Four Enterococcus cecorum Strains Isolated from American Black Vultures (Coragyps atratus) and Harris's Hawk (Parabuteo unicinctus) in Rio de Janeiro, Brazil.

We report the draft genome sequences of four Enterococcus cecorum strains obtained from cloacal swab specimens collected from three healthy captive wild birds (two Coragyps atratus and one Parabuteo unicinctus) in Rio de Janeiro, Brazil. The genome sizes ranged from 2.38 to 2.55 Mb.

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil.

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.

Draft Genome Sequences of Two Commensal Enterococcus faecalis Strains Isolated from American Black Vultures (Coragyps atratus) in Brazil.

We report the draft genome sequences of two commensal Enterococcus faecalis strains (designated Ca-2 and Ca-18) recovered from the cloacae of two healthy American black vultures (Coragyps atratus) in Rio de Janeiro, Brazil. The strains were found to carry a variety of antimicrobial resistance and virulence-associated genes.

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.

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.

First report of the bla(OXA-58) gene in a clinical isolate of Acinetobacter baumannii in Rio de Janeiro, Brazil.

Carbapenemase production is an important mechanism of carbapenem resistance among nonfermentative Gram-negative isolates. This study aimed to report the detection of bla(OXA-58) gene in multiresistant clinical isolates of Acinetobacter baumannii recovered from inpatients in a public hospital. Polymerase chain reaction tests were performed to detect the bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like) and bla(OXA-51-like) genes. The bla(OXA-58) and bla(OXA-23) genes were detected in one and three isolates, respectively. Sequencing of the bla(OXA-58-like) amplicon revealed 100% identity with the A. baumannii bla(OXA-58) gene listed in the GenBank database. This is the first report of an OXA-58-producing A. baumannii isolate in Rio de Janeiro, Brazil.

Characterization of Enterococcus faecium E86 bacteriocins and their inhibition properties against Listeria monocytogenes and vancomycin-resistant Enterococcus.

In the present scenario of a major demand for new compounds with antimicrobial activity, bacteriocin and bacteriocin-like inhibitory substances (BLIS) are promising tools against deteriorating and pathogenic microorganisms, thus having potential applications in both the food industry and infectious disease control. In the present report, we describe the genetic and phenotypic characteristics of BLIS produced by Enterococcus faecium E86, a strain previously isolated and sequenced by our group, focusing on the structural genes of two bacteriocins identified: enterocin TW21 and enterocin P. Transcription of all four genes associated with the biosynthesis and immunity of enterocin P and enterocin TW21 were confirmed by RT-PCR. However, Sanger sequencing confirmed a truncation of the structural gene of enterocin TW21 due to one base pair deletion (A/T). Thus, although E. faecium E86 was shown to carry two bacteriocinogenic gene clusters, only one cluster encodes a functional bacteriocin, enterocin P. Enterocin P was able to inhibit different strains of Listeria monocytogenes and vancomycin-resistant enterococci (both Enterococcus faecalis and Enterococcus faecium), showing intense bacteriolytic activity, in most cases.

Unusual finding of the human-adapted hypervirulent serotype III/ST17 clone in a historical bovine Group B Streptococcus isolate from Brazil.

Group B Streptococcus (GBS) is a leading cause of human neonatal infections and bovine mastitis. We report here the unusual finding of the human-adapted hypervirulent serotype III/ST17 clone in a bovine GBS isolated in 1987 in Brazil. This isolate shared several phenotypic and genotypic characteristics with serotype III/ST17 strains obtained from human sources, including PFGE pattern, pilus genes, lactose fermentation, DNase activity, and antimicrobial susceptibility profile, highlighting the importance of continued tracking of GBS in the One Health scope. The study brings new evidence for the potential interspecies transmission and sheds new light into evolution aspects of the pathogen Group B Streptococcus (GBS) by reporting the occurrence of an ancient bovine GBS isolate belonging to a variant currently known to be exclusively found in human hosts.

Molecular characterization of uropathogenic and diarrheagenic Escherichia coli pathotypes.

In this study diarrheagenic and uropathogenic Escherichia coli (UPEC) strains were comparatively characterized according to serotype, hemolytic activity, protein polymorphism among housekeeping enzymes, phylogenetic group and urovirulence genes. Intra-serogroup/serotype variations were observed. Hemolytic activity was detected in 100%, 93%, 67% and 39% of UPEC, EAEC, EPEC and ETEC strains, respectively. The alpha-hemolytic phenotype was observed in all pathogenic groups while beta-hemolytic phenotype was less frequent. PCR phylotyping revealed higher prevalence of diarrheagenic E. coli in groups A and D while uropathogenic strains were mainly found in subgroup B2. Amplification assays revealed that 74%, 45% and 22% of UPEC, EAEC and EPEC strains, respectively, carried at least one of the urovirulence sequences. The molecular typing system revealed a pathotype-specific clonal group distribution and showed a closer relationship between the EAEC and UPEC. Additionally, the occurrence of urovirulence traits, especially those related to iron acquisition, was more frequent among EAEC and UPEC than among the other E. coli pathotypes. This observation is of special value considering that the EAEC pathotype constitutes an emerging group of enteropathogens, particularly, in developing countries, and information on their pathogenic and phylogenetic characteristics is still scarce.

Prevalence of PspA families and pilus islets among Streptococcus pneumoniae colonizing children before and after universal use of pneumococcal conjugate vaccines in Brazil.

In 2010, the 10-valent (PCV10) and 13-valent (PCV13) pneumococcal conjugate vaccines were introduced in Brazil to immunize children, resulting in serotype replacement. We analyzed 253 carriage isolates recovered from children aged <6 years in Brazil, including 124 and 129 isolates from the pre-PCV10/13 (December 2009-July 2010) and post-PCV10/13 (September-December 2014) periods, respectively, to investigate the prevalence of PspA families and pilus islets, potential vaccine candidates. Serotypes and resistance profiles were previously characterized. We used PCR to type PspA families (Fam1-3) and pilus islets (PI-1 and PI-2). We identified the PspA family of 130 (51.4%) isolates. PspA families 1, 2, and 3 were identified in 12.2%, 38.7%, and 0.4% of the isolates, respectively. Eighteen (58.1%) Fam1 isolates were serogroup 6. Nine (81.8%) of 11 serotype 14 isolates were Fam2. Fam1 isolates resistant to penicillin (50%), erythromycin (43.7%), clindamycin (31.2%), and chloramphenicol (6.2%) were only found after PCV10/13 introduction. Resistance among Fam2 isolates was higher in the post-PCV10/13 period to erythromycin (1.8% vs. 18.6%), clindamycin (0 vs. 13.9%), and tetracycline (10.9% vs. 16.3%). PI-I was detected in 42 (16.6%) isolates. Fourteen (56%) of 25 serotype 15B/C and nine (81.8%) of 11 serotype 14 isolates had PI-1 (p < 0.01). Eight (3.2%) isolates had PI-2, and six (75%) were serogroup 19. Five (2%) serogroup 19 isolates had both PI-1 and PI-2. We found associations between serogroups/serotypes, PspA families, and pilus islets, but distribution of PspA families and pilus islets was similar in both periods. After universal vaccination, we observed higher antimicrobial resistance frequencies, regardless PspA or pilus types.

Expression of VanA-type vancomycin resistance in a clinical isolate of Enterococcus faecium showing insertion of IS19 in the vanS gene.

The characteristics of an unusual clinical isolate of Enterococcus faecium (CL-6729) showing insertion of IS19 (also known as ISEfm1) in the vanS gene while maintaining a constitutive VanA phenotype are described. This isolate was obtained from a hospital-acquired urinary tract infection, showed multidrug resistance by antimicrobial susceptibility testing, and belongs to ST78 based on multilocus sequence typing (MLST). Except for the vanS gene, all the other genes of the vanA gene cluster were intact according to conventional PCR, overlapping PCR and genome sequencing. By quantitative reverse transcription PCR (RT-qPCR), the isolate showed similar expression of the vanA, vanR and vanS genes in the presence and absence of vancomycin. The results suggest that insertion of IS19 in the vanS gene may be associated with constitutive expression of resistance to vancomycin in clinical isolate CL-6729, either by not impairing VanS activity or by inducing the emergence of another pathway that acts on vanA expression, which still needs to be fully investigated.

CRISPR elements and their association with antimicrobial resistance and virulence genes among vancomycin-resistant and vancomycin-susceptible enterococci recovered from human and food sources.

We aimed to investigate the occurrence of CRISPR elements in the genomes of vancomycin-resistant (VRE) and vancomycin-susceptible (VSE) enterococci and their association with the presence of antimicrobial resistance and virulence genes. We analyzed 180 isolates, including 91 VRE and 89 VSE. Isolates were identified by PCR or MALDI-TOF. Antimicrobial susceptibility and MICs for vancomycin were determined by the disk-diffusion method and E-test®, respectively. The presence of resistance and virulence genes, as well as CRISPR elements, was investigated by PCR. We identified 95 (53%) E. faecalis, 78 (43%) E. faecium, five (2.8%) E. gallinarum, and one (0.6% each) E. casseliflavus and E. durans. The highest and the lowest non-susceptibility frequencies were observed for erythromycin (n = 152; 84.4%) and fosfomycin (n = 5; 2.8%), respectively. Most erythromycin-resistant isolates had the erm(B) gene (106/152; 69.7%). Of 118 (65.6%) isolates with high-level resistance to aminoglycoside, 69 (58.5%) had at least one aminoglycoside resistance gene, mostly ant(6)-Ia and aac(6')-Ie + aph(2″)-Ia. We found at least one virulence gene among 135 (75%) isolates, mostly gelE (79/180; 43.9%). Ninety-two (51.1%) isolates had at least one CRISPR element, especially CRISPR3 (62/92; 67.4%). CRISPR elements were more common among E. faecalis, in which we observed a relationship between the absence of CRISPR and the presence of the vanA resistance gene, and the hyl and esp virulence genes. Among VRE. faecium, a relationship was found between the absence of CRISPR and the hyl gene. In conclusion, we found evident associations between the lack of CRISPR elements with species, multidrug resistance, and major resistance- and virulence-associated genes.