Whole-genome analysis of haemophilus influenzae invasive strains isolated from Campinas state University hospital. An epidemiological approach 2012 - 2019 and ancestor strains

Abstract Thirteen Haemophylus influenzae invasive strains isolated from patients at Clinical Hospital of State University of Campinas, from May 2013 through August 2019, was submitted to Illumina genome sequencing HiSeq platform. Further in silico analysis of serogroup and Multi Locus Sequence Typing (MLST) from whole DNA sequencing had demonstrated the actual clonal distribution in the Campinas Metropolitan region. Thus, results showed the existence of a new ST Haemophilus influenzae found in the Brazilian territory and an increase of strains belonging to serogroup a (three strains also belonging to ST23). In conclusion, we observed an increase of non-typable H. influenzae (NTHi) and a strain involved in invasive diseases in the Campinas - São Paulo region after frequent detection of those serotypes and genotypes in other Brazilian regions.

Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.

Opsonization and transformation: effects of anticapsular sera on the DNA transfer in Neisseria meningitidis

The capsular switching process indicates the action of specific capsular antibodies on the meningococcal strains adaptation. Different antibodies were employed for assessing the effect of opsonization on the transformation of Neisseria meningitidis serogroups C and W135. These analyses showed the blocking action of the specific capsular antibodies on the meningococcal transformation capacity. Thus, the blocking effect of these antibodies on N. meningitidis transformation process was demonstrated. This effect could be involved in the capsular switching process and the found data might open new subjects for scientific exploratio.

Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in two metropolitan areas of São Paulo State, southeast Brazil

One hundred and fifty-one methicillin-resistant z (MRSA) strains have been isolated from patients admitted in tertiary care hospitals in two metropolitan areas (Campinas City and Ribeirão Preto City) in the southeast region of Brazil and analyzed through PCR-based techniques [(PCR amplification of spa, coa, and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] and further restriction fragment typing of coa and of housekeeping genes. The heterogeneity of spa gene was determined directly by agarose gel electrophoresis migration. The results obtained indicate the existence of three (A, B, C) main clusters. Since the strain distribution in these three clusters is much characteristic, it denotes the existence of three main clones. All strains isolated in Campinas were grouped in clusters A and B, while most of the strains isolated in Ribeirão Preto were grouped in cluster C. This distribution denotes the existence of different founder strains that undergo independent genetic variability. The strains considered representative of the Brazilian Epidemic Clone (BEC) were categorized as cluster A. These results indicate a possible higher variability among Brazilian MRSA strains than currently described and indicate that the techniques herein used can be used as an alternative to Pulsed Field Gel Electrophoresis (PFGE).

Pathogenic and opportunistic respiratory bacteria-induced apoptosis

Several pathogenic or opportunistic bacteria have the ability to either induce or inhibit host cell apoptosis. The capacity to modulate cell pathways that result in the induction or delay of host cell apoptosis is considered to be an important bacterial virulence mechanism. These processes could be mediated by different host cell signaling pathways that are subverted by the bacteria. Pathogens are able to activate apoptotic proteins, such as caspases, or inactivate anti-apoptotic proteins, such as NFkB and the MAPKKs, or even up-regulate the endogenous receptor/ligand system that induces apoptosis, generally when the bacteria are bound to the host cell surface. The bacteria-induced apoptotic or anti-apoptotic processes are often related with the fact that the bacteria acquire the ability to reach the host tissues. However, apoptosis is also considered to be a host defense mechanism against infectious agents. Thus, the apoptosis phenomenon plays a central role in host-pathogen interactions.

Ribotyping, biotyping and capsular typing of Haemophilus influenzae strains isolated from patients in Campinas, southeast Brazil

Forty-five Haemophilus influenzae strains isolated from patients were characterized based on biochemical characteristics. Their capsular types were determined by polymerase chain reaction (PCR); they were compared, using two molecular methods [ribotyping with a specific DNA probe amplified from the 16S rDNA region from H. influenzae and through restriction fragment length polymorphism (RLFP) of an amplified 16S DNA region]. The strains were better discriminated by the ribotyping technique that used the 16S probe and by the combination of both techniques. Biotypes I and IV were the most common, followed by biotypes VI, VIII and III. Biotypes II and VII were not found. Most of the capsular samples were nontypable (89 percent), with capsular types a and b found in 2 and 9 percent of the samples, respectively. We concluded that there is a very close genetic identity among pathogenic and non-pathogenic strains.

Research on staphylococcus spp in biofilm formation in water pipes and sensibility to antibiotics

Water from dental equipment presents risks for surgeon-dentists as well as for patients because it might work as a means of dissemination/ transmission of microorganisms. The objective of this study was to verify the quality of the water used in dental equipment by means of microbiological analysis, accomplishing the count of Staphylococcus spp. There have been collected 160 samples of water from reservoirs, taps used for hand washing, air-water syringes, and high-speed handpieces, in 40 dental offices in the city of Barretos, São Paulo. The rules concerning bacteriological analysis in cfu/mL from Standard Methods for the Examination of Water and Wastewater have been followed. The analysis of the results has made it possible to verify that out of the total of samples, 28% did not meet the standards of potability established by the American Dental Association. Regarding the origin of analyzed S. aureus, the most contaminated sites were high-speed handpieces in private offices (76%) and in dental care plan offices (71%), followed by air-water syringe in dental care plan offices (64%). For S. epidermidis samples, the most contaminated sites were high-speed handpieces in SUS (Brazilian Government Health System) dental offices (22%) and in dental care plan offices (14%). The most contaminated sites were dental offices that saw patients under dental care plans. Concerning tested antibiotics, the ones that presented better results as to sensibility to strain S. epidermidis were vancomycin and ciprofloxacin (100%) and, as to sensibility to strain S. aureus, it was ciprofloxacin (97%).

Typing of avian pathogenic Escherichia coli strains by REP-PCR

A técnica de REP (Repetitive extragenic palindrome)-PCR foi utilizada para avaliar a variabilidade genética de 49 amostras de Escherichia coli patogênicas para aves (APEC), isoladas de aves de corte (frangos) em diferentes surtos de septicemia (n=24), síndrome da cabeça inchada (n=14) e onfalite (n=11). Trinta amostras comensais, isoladas de frangos sem sinais de doença, foram utilizadas como controle. A análise do perfil eletroforético obtido por reação de REP-PCR utilizando DNA purificado das amostras evidenciou a amplificação de 0 a 15 bandas de DNA com pesos moleculares variando entre 100 pb e 6.1 Kb. A análise deste padrão permitiu a construção de um dendrograma demonstrando o agrupamento das 79 amostras em 49 perfis distintos. Embora a técnica de REP-PCR tenha apresentado grande poder discriminatório, as amostras patogênicas e não patogênicas não foram discriminadas entre si assim como não foi observado o agrupamento de amostras causadoras do mesmo tipo de doença. Por outro lado, demonstramos recentemente que outras técnicas tais como ERIC-PCR e a análise de isoenzimas foram eficientes quando utilizadas para esta mesma finalidade. Concluindo, REP-PCR parece não ser uma técnica eficiente e universal para discriminar entre amostras APEC. Porém, a estrutura clonal populacional obtida com o uso de REP-PCR não deve ser desprezada, particularmente se considerarmos que os mecanismos de patogenicidade de APEC ainda não são completamente conhecidos.

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Bacteria-induced apoptosis: an approach to bacterial pathogenesis

Several pathogenic or opportunistic bacteria can induce or inhibit host cell apoptosis. The modulation of cellular pathways that results in the induction or delay of host cell apoptosis is an important mechanism of bacterial virulence. These processes can be mediated by various host cell signaling pathways that are subverted by the bacteria. Pathogens can activate apoptotic proteins such as caspases, inactivate anti-apoptotic proteins such as NFêB and mitogen-activated protein kinases, or up-regulate the endogenous receptor/ligand system that induces apoptosis, generally when the bacteria are bound to the host cell surface. Bacteria-induced apoptotic or anti-apoptotic processes are often related to the ability of the bacteria to reach the host tissues. However, since apoptosis is also involved in host defense mechanisms against infectious agents, this phenomenon apparently plays a central role in host-pathogen interactions.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits