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

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.

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.

Analysis of potential virulence genes and competence to transformation in Haemophilus influenzae biotype aegyptius associated with Brazilian Purpuric Fever.

Brazilian Purpuric Fever (BPF) is a hemorrhagic pediatric illness caused by Haemophilus influenzae biogroup aegyptius (Hae), a bacterium that was formerly associated with self-limited purulent conjunctivitis. BPF is assumed to be eradicated. However, the virulence mechanisms inherent to Hae strains associated with BPF is still a mystery and deficient in studies. Here, we aim to analyze the role of the autotransporter genes related to adherence and colonization las, tabA1, and hadA genes through RT-qPCR expression profiling and knockout mutants. Relative quantification by real-time PCR after infection in human cells and infant rat model suggests that las was initially downregulated probably duo to immune evasion, tabA1, and hadA were overexpressed in general, suggesting an active role of TabA1 and HadA1 adhesins in Hae in vitro and in vivo. Transformation attempts were unsuccessful despite the use of multiple technical approaches and in silico analysis revealed that Hae lacks genes related to competence in Haemophilus, which could be part of the elucidation of the difficulty of genetically manipulating Hae strains.

Preparation of Thermosensitive Gel for Controlled Release of Levofloxacin and Their Application in the Treatment of Multidrug-Resistant Bacteria.

Levofloxacin is a synthetic broad-spectrum antibacterial agent for oral or intravenous administration. Chemically, levofloxacin is the levorotatory isomer (L-isomer) of racemate ofloxacin, a fluoroquinolone antibacterial agent. Quinolone derivatives rapidly and specifically inhibit the synthesis of bacterial DNA. Levofloxacin has in vitro activity against a broad range of aerobic and anaerobic Gram-positive and Gram-negative bacteria. However, formulation of combined poloxamers thermoregulated (as Pluronic® F127) and levofloxacin for use in multiresistant bacterial treatment were poorly described in the current literature. Thus, the aim of the present work is to characterize poloxamers for levofloxacin controlled release and their use in the treatment of multidrug bacterial resistance. Micelles were produced in colloidal dispersions, with a diameter between 5 and 100 nm, which form spontaneously from amphiphilic molecules under certain conditions as concentration and temperature. Encapsulation of levofloxacin into nanospheres showed efficiency and enhancement of antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae when compared with only levofloxacin. Furthermore, all formulations were not cytotoxic for NIH/3T3 cell lineage. In conclusion, poloxamers combined with levofloxacin have shown promising results, better than alone, decreasing the minimal inhibitory concentration of the studied bacterial multiresistance strains. In the future, this new formulation will be used after being tested in animal models in patients with resistant bacterial strains.

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.

Haemophilus influenzae porine ompP2 gene transfer mediated by graphene oxide nanoparticles with effects on transformation process and virulence bacterial capacity.

BACKGROUND: H. influenzae is a natural competent bacterium that can uptake DNA from the environment and recombine into bacterial genome. The outbreaks of Brazilian purpuric fever, heavily polluted areas of a different H. influenzae biogroup - aegyptius - as well as gene transference between Neisseria meningitis make the transformation process an important evolutionary factor. This work studied the horizontal transference of the ompP2 gene from a multiresistant strain of H. influenzae 07 (NTHi), under the influence of graphene oxide nanoparticles in order to mimic an atmosphere rich in suspended particles and this way verify if the CFU transformants number was increased. MATERIAL AND METHODS: In this article the gene ompP2 was transformed into different strains of H. influenzae mediated or not by graphene oxide nanoparticles in suspension, followed by the adhesion tests in Hec-1B (human endometrium adenocarcinoma) and A549 (pulmonary epithelial carcinoma) cells lines. The transformation frequency and the adhesion capacity were determined in all the mutants to which the ompP2 gene was transferred and compared to their wild type strains. RESULTS: The nanoparticles increased the transformation ratio of one particular strain isolated from a pneumonia case. The adhesion patterns to A549 and Hec1b cell lines of these mutated bacteria has their capacity increased when compared to the wild type. CONCLUSIONS: Graphene oxide nanoparticles aid the transformation process, helping to increase the number of CFUs, and the mutants generated with the ompP2 gene from a H. influenzae resistant strain not only present a chloramphenicol resistance but also have an increased adherence patterns in A549 and Hec1B cell lines.

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.

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.

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.