Trends in serotype distribution and antimicrobial susceptibility pattern of invasive Haemophilus influenzae isolates from Brazil, 2009-2021.

INTRODUCTION: 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. METHODOLOGY: This study analyzed 1437 invasive Hi isolates in Brazil over 13 years, determining capsular serotypes, antimicrobial susceptibility, and genetic relatedness through multilocus sequence typing. RESULTS: The primary source of isolation for these invasive H. influenzae isolates was blood (54.4%), followed by cerebrospinal fluid (37.1%) and lung specimens (8.5%), respectively. Consequently, bacteremia (47%) was the most common clinical presentation, followed by meningitis (39.6%) and pneumonia (13.4%). Non-encapsulated Hi (NTHi) predominated among the isolates (51.4%), along with serotype a (22%) and serotype b (21.5%) among the encapsulated isolates. The majority of the encapsulated isolates were isolated from children under 14 years of age (76.7%), while NTHi isolates were identified in patients older than 15 years, particularly those ≥ 60 years old (40%). Ampicillin resistance was observed in 17.1% of cases, displaying ß-lactamase production as the principal resistance mechanism. MLST revealed a diverse NTHi population, whereas the encapsulated isolates presented a clonal structure. CONCLUSION: This study describes the prevalence of NTHi isolates circulating in Brazil after two decades of the Hib vaccine immunization program. Continuous universal surveillance is crucial for implementing prompt public health measures to prevent and control invasive Hi disease and monitor changes in antibiotic resistance profiles.

Rapid response of a public health reference laboratory to the COVID-19 pandemic.

Introduction. Brazil was one of the most affected countries by the COVID-19 pandemic. Instituto Adolfo Lutz (IAL) is the reference laboratory for COVID-19 in São Paulo, the most populous state in Brazil. In April 2020, a secondary diagnostic pole named IAL-2 was created to enhance IAL's capacity for COVID-19 diagnosis.Hypothesis/Gap Statement. Public health laboratories must be prepared to rapidly respond to emerging epidemics or pandemics.Aim. To describe the design of IAL-2 and correlate the results of RT-qPCR tests for COVID-19 with secondary data on suspected cases of SARS-CoV-2 infection in the São Paulo state.Methodology. This is a retrospective study based on the analysis of secondary data from patients suspected of infection by SARS-CoV-2 whose clinical samples were submitted to real-time PCR after reverse transcription (RT-qPCR) at IAL-2, between 1 April 2020 and 8 March 2022. RT-qPCR Ct results of the different kits used were also analysed.Results. IAL-2 was implemented in April 2020, just over a month after the detection of the first COVID-19 case in Brazil. The laboratory performed 304,250 RT-qPCR tests during the study period, of which 98 319 (32.3 %) were positive, 205827 (67.7 %) negative, and 104 (0.03 %) inconclusive for SARS-CoV-2. RT-qPCR Ct values≤30 for E/N genes of SARS-CoV-2 were presented by 79.7 % of all the samples included in the study.Conclusion. IAL was able to rapidly implement a new laboratory structure to support the processing of an enormous number of samples for diagnosis of COVID-19, outlining strategies to carry out work with quality, using different RT-qPCR protocols.

Molecular characterization of persistent subclinical mastitis-causing Staphylococcus aureus from dairy farms.

The study aimed to evaluate the genetic diversity of Staphylococcus aureus causing subclinical mastitis (SM) isolated from dairy cows and to assess the effect of the infection status (transient vs. persistent) on the milk and component yield. A total of six dairy farms in São Paulo state were used for the selection of cows with SM caused by S. aureus. S. aureus strains (n = 56) obtained from three biweekly aseptic mammary quarter milk samplings (n = 1140 from 95 cows) were subjected to MALDI-TOF MS analysis for species confirmation and further PFGE analysis. Intramammary infections (IMI) caused by S. aureus were categorized as transient (T: when only one out of 3 milk samplings had positive isolation of any pulsotype) or persistent (P: when two (P2) or three (P3) milk samplings had positive isolation of identical pulsotype over the consecutive episodes of SM. The SmaI macrorestriction fragment profiles of 56 S. aureus isolates showed a dominant S. aureus clonal pattern (PFGE type A; n = 50; 89.3%) within and among the herds. The SM-causing S. aureus represented a reduction of quarter milk yield of 26.2% in transient and 54.8% in persistent cases as well as a reduction of total solid yield of 38.1% and 49.4%, respectively, when compared with the healthy control quarters. Overall, the greater chance of S. aureus to be persistent is when a dominant clonal pattern is present in the herd which consequently may be associated with the cause of accentuated milk loss.

A new reduced chalcone-derivative affects the membrane permeability and electric potential of multidrug-resistant Enterococcusfaecalis.

The emergence and spread of multidrug-resistant (MDR) enterococci and other Gram-positive bacteria represents a severe problem due to the lack of effective therapeutic alternatives. Natural products have long been an important source of new antibacterial scaffolds and can play a key role in the current antibiotic crisis. Enterococci are predominantly non-pathogenic gastrointestinal commensal bacteria, but among them, Enterococcus faecalis and Enterococcus faecium represent the species that account for most clinically relevant infections. The emergence of MDR enterococci has reduced the available antibiotic treatment options and highlights the need to develop new antimicrobial compounds. In the search for new hit compounds against MDR Enterococcus spp., natural-derived compounds represent inspiring scaffolds for drug design studies. In this work, the antimicrobial activity of a fully synthetic chalcone derivative (r4MB) was determined on a clinical panel of 34 MDR Gram-positive bacteria, mostly constituted by E. faecalis and E. faecium, along with Staphylococcus spp., amongst others. Compound r4MB showed activity against 100% of the tested strains, with the minimum inhibitory concentration (MIC) in the range of 5-20 µM. The lethal action of the compound was evaluated using different fluorescent-based assays. The compound showed a time-dependent permeabilisation of the membrane of a vancomycin-resistant E. faecalis, detected by the fluorescent probe SYTOX Green, and digital fluorescent microscopy corroborated the spectrofluorimetric analysis within 6 min of incubation. Flow cytometry analysis of the membrane electric potential demonstrated a significant depolarization, confirming the target of the compound towards the bacterial membrane. No cytotoxic haemolysis was observed with mammalian erythrocytes, and a 99% cytotoxic concentration of 118 µM on NCTC cells demonstrated a promising antimicrobial selectivity. In silico studies using SwissADME and ADMETLabs servers suggest that compound r4MB displayed adequate ADME properties, with no alerts for pan-assay interference compounds (PAINS). Future hit-to-lead optimization of this chalcone derivative can contribute to developing a more potent derivative against infections caused by MDR enterococci.

Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis isolates are uncommon in non-clinical sources.

This study aimed to investigate whether penicillin-resistant, ampicillin-susceptible E. faecalis (PRASEF) isolates are disseminated in non-clinical sources, and to compare the molecular characteristics and antimicrobial resistance (AMR) profile of clinical and non-clinical E. faecalis isolates. Non-clinical samples (n = 280) were collected and 101 E. faecalis isolates were recovered from food (n = 18), faeces of healthy animals (n = 24), water (n = 28) and sewage (n = 31). PRASEF (n = 68) and penicillin-susceptible, ampicillin-susceptible E. faecalis (n = 77) isolates of clinical origin were also evaluated. A significant variety of AMR profiles was observed among non-clinical isolates according to the source. No food isolate exhibited a multidrug resistance (MDR) phenotype different from those of isolates from animal faeces (50.0%) and sewage (38.7%). Overall, the MDR phenotype was more frequent among clinical (56.6%) than non-clinical isolates (22.8%) (p < 0.01). Non-clinical PRASEF isolates (n = 3) were only recovered from hospital sewage. Note that representative clinical and non-clinical PRASEF isolates were grouped in pulsotype A, and belonged to CC9 (clonal complex). In conclusion, E. faecalis isolates exhibiting the unusual penicillin-resistant but ampicillin-susceptible phenotype appeared to be restricted to the hospital environment. Our findings highlight the ability of PRASEF isolates to survive in sewage, which could enable these hospital-adapted lineages to spread to new ecological niches.

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in a Brazilian elderly cohort.

We aimed to investigate the nasopharyngeal colonization (NPC) by Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in the elderly population and to assess the demographic factors associated with NPC. This was an observational cohort study in which outpatients aged ≥60 years were enrolled from April to August 2017, with a follow-up visit from September through December 2017. Nasopharyngeal (NP) swabs were collected, bacteria were detected and isolated, and isolates were subjected to phenotypic and molecular characterization using standard microbiological techniques. At enrolment, the rates of S. aureus, methicillin-resistant S. aureus (MRSA), H. influenzae, and S. pneumoniae among 776 elderly outpatients were 15.9%, 2.3%, 2.5%, and 2.2%, respectively. Toxin production was detected in 21.1% of methicillin-susceptible S. aureus, and three SCCmec types were identified: II/IIb, IVa, and VI. At the follow-up visit, all carriage rates were similar (p > 0.05) to the rates at enrolment. Most of S. pneumoniae serotypes were not included in pneumococcal conjugate vaccines (PCVs), except for 7F, 3, and 19A. All strains of H. influenzae were non-typeable. Previous use of antibiotics and 23-valent pneumococcal polysaccharide vaccination (p < 0.05) were risk factors for S. aureus and MRSA carriage; S. aureus colonization was also associated with chronic kidney disease (p = 0.021). S. pneumoniae carriage was associated with male gender (p = 0.032) and an absence of diabetes (p = 0.034), while not receiving an influenza vaccine (p = 0.049) and chronic obstructive pulmonary disease (p = 0.031) were risk factors for H. influenzae colonization. The frailty of study participants was not associated with colonization status. We found a higher S. aureus carriage rate compared with the S. pneumoniae- and H. influenzae-carriage rates in a well-attended population in a geriatric outpatient clinic. This is one of the few studies conducted in Brazil that can support future colonization studies among elderly individuals.

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil.

The aim of the present study was to assess the prevalence of Haemophilus influenzae type b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzaetype b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

Prevalence and antimicrobial susceptibility profile of Enterococcus spp isolated from frozen chicken carcasses

Prevalence and antimicrobial susceptibility of Enterococcus spp. were evaluated in 360 frozen unse asoned chicken carcasses samples collected from September 2004 to June 2006 from the retail stores in São Paulo State, Brazil. Enterococcus spp. was isolated from all analyzed samples, and 1,332 strains were identified from them. Among the ten identified species, the predominance of E. faecalis, E. gallinarum, E.casseliflavus and E. faecium was occurred. All of the Enterococci strains showed some degree of resistanceto the nine antimicrobials utilized in the study. The percentages of antimicrobial resistance were: 89.2% for tetracycline, 91.4% for quinupristin-dalfopristin, 83.5% for erythromycin, 65% for ciprofloxacin, 55.4% for chloramphenicol, 6.5% for linezolid, 2.3% for vancomycin, 2.3% for teicoplanin and 0.2% for ampicillin. The occurrence of the high level resistance to amyno glicosides (HLR-A) was detected in 57.4% of the isolates. E. faecalis and E. faecium species, which are considered as important agents in nosocomial infections, showed resistance to eight and seven antibiotics, respectively.

Carriage of Haemophilus influenzae among Brazilian children attending day care centers in the era of widespread Hib vaccination.

Haemophilus influenzae type b vaccine was introduced into the Immunization Program of Brazil in 1999 and no study has evaluated the impact of Hib vaccination in H. influenzae carriage so far. In June 2010, Brazil introduced the 10-valent pneumococcal nontypeable H. influenzae (NTHi) conjugate vaccine (PHiD-CV). We investigated the prevalence of encapsulated H. influenzae and NTHi isolates in nasopharyngeal samples of 1192 children attending day-care centers in Goiânia, central Brazil. H. influenzae carriage rate was 32.1% and 38.4% of them carried ß-lactamase TEM-1 gene. Serotype f (4.6%) was the most frequent encapsulated isolate, type b was recovered in only 0.7% and carriage rate of NTHi was 23.3%. Recurrent acute otitis media and NTHi were independently associated with colonization by ß-lactamase producing H. influenzae. Changes in frequency of H. influenzae carriage isolates should be carefully monitored to assess the impact of the PHiD-CV on NTHi carriage in young children.

Molecular characterization of enterococci harboring genotype and phenotype incongruence related to glycopeptide resistance isolated in Brazilian hospitals.

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.

Molecular assessment of invasive Streptococcus pneumoniae serotype 1 in Brazil: evidence of clonal replacement.

In Brazil, serotype 1 Streptococcus pneumoniae is one of the most prevalent causes of severe infection. This study investigated the genetic relatedness of 134 serotype 1 isolates obtained from invasive diseases during the period 1977-2005. Molecular typing by PFGE revealed two major lineages using visual inspection and computer analysis. Type A comprised 94 isolates (70.2 %) with four subtypes, whereas type B comprised 40 isolates (29.8 %) with eight subtypes. Subtype A3, the most frequent genotype, accounting for 65 % of the total isolates, was identified as a representative of clone Sweden(1)-40 (ST304). Type B was predominant in the period 1977-1988. In contrast, an increase in the type A lineage was detected from 1990 in Brazil, significantly associated with isolates recovered from pneumonia cases and from young patients. This study clearly established a temporal switch between two lineages of S. pneumoniae serotype 1 in Brazil, with a wide dispersion of clone Sweden(1)-40 in recent years.

Molecular characterization of enterococci harboring genotype and phenotype incongruence related to glycopeptide resistance isolated in Brazilian hospitals

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.

Emergence of VanB phenotype-vanA genotype in vancomycin-resistant enterococci in Brazilian hospital

No Brasil, enterococos resistente à vancomicina (VRE) têm sido descritos como patógenos hospitalares, desde 1998. Durante um monitoramento de VRE em um hospital, foram detectadas duas cepas de Enterococcus faecalis com genótipo vanA, e sensibilidade à teicoplanina. Este é o primeiro relato do isolamento de enterococo fenótipo VanB e genótipo vanA de amostra clínica no Brasil.

Increase in numbers of beta-lactam-resistant invasive Streptococcus pneumoniae in Brazil and the impact of conjugate vaccine coverage.

A comprehensive investigation of invasive Streptococcus pneumoniae was carried out in Brazil as part of the programme of the national epidemiological surveillance system. The investigation provided data on the trends of resistance to antimicrobial agents. A total of 6470 isolates of S. pneumoniae collected in the country from 1993 to 2004 were tested. During this period of time, the number of penicillin-resistant strains rose from 10.2 to 27.9%. The proportions of intermediate and high-level resistant strains in 1993, which were 9.1 and 1.1%, respectively, rose to 22.0 and 5.9% in 2004. Geometric mean MICs for penicillin increased after the year 2000, to 0.19 microg ml(-1) in 2004; most of these isolates were from patients with pneumonia and from children under 5 years old, and belonged to serotype 14. There was a significant increase in the number of isolates belonging to serotypes included in the 7-valent conjugate vaccine from children under 5 years old: from 48.6% in 1993 to 69.6% in 2004, mainly related to an increase in the frequency of serotype 14 isolates. From 2000 to 2004, meningitis isolates showed higher resistance rates to cefotaxime (2.6%) compared to non-meningitis isolates (0.7%); percentages of isolates resistant to trimethoprim-sulfamethoxazole, tetracycline, erythromycin, chloramphenicol and rifampicin were 65, 14.6, 6.2, 1.3 and 0.7 %, respectively. No levoflaxin resistance was observed. Multidrug resistance was identified in 4.6% of isolates, of which 3.8% were resistant to three classes, 0.7% to four classes and 0.1% to five classes of antimicrobial agent. The study provides valuable information that may support empirical antimicrobial therapy for severe S. pneumoniae infections in Brazil, and emphasizes the need for conjugate pneumococcal vaccination.