Agent Of Pneumonia Research Articles

A genetic and virulence characterization of Brazilian strains of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae (M. hyopneumoniae) is considered the primary causative agent of porcine enzootic pneumonia (EP), a chronic contagious respiratory disease that causes economic losses. Obtaining new pathogenic isolates and studying the genome and virulence factors are necessary. This study performed a complete sequencing analysis of two Brazilian strains, UFV01 and UFV02, aiming to characterize the isolates in terms of the virulence factors and sequence type. The complete genome analysis revealed the main virulence genes (mhp385, mhp271, MHP_RS03455, p102, p97, p216, MHP_RS00555, mhp107) and ST-123, the presence of three toxin-related genes (tlyC, PLDc_2 and hcnC), and some genetic groups specific to these two isolates. Subsequently, the pathogenicity of the isolates was evaluated via an experimental infection conducted in a swine model. The study was divided into three groups, namely a negative control group (n = 4) and two test groups (n = 8), totaling 20 animals. They were challenged at 35 days of age with 107 CCU (Color Changing Units) M. hyopneumoniae via the intratracheal route. The UFV01 group showed earlier and higher seroconversion (IgG) (100%), while only 50% of the UFV02 group seroconverted. The same trend was observed when analyzing the presence of IgA in the bronchoalveolar lavage fluid (BALF) at 35 days post-infection (dpi). The UFV01 group had a mean macroscopic lesion score of 11.75% at 35 dpi, while UFV02 had 3.125%. Microscopic lesions were more severe in the UFV01 group. Based on laryngeal swab samples evaluated by qPCR, and the detection began at 14 days. The UFV01 group showed 75% positivity at 14 dpi. The UFV02 group also started excreting at 14 dpi, with a positivity rate of 37.5%. The results indicate that the UFV01 isolate exhibits higher virulence than UFV02. These findings may aid in developing new vaccines and diagnostic kits and establishing experimental models for testing.

Management and Control of Legionella pneumophila Nosocomial Outbreak Risk in a Tertiary Level Pediatric Hospital

Abstract Background Legionnaires' disease is a rare cause of pneumonia in the pediatric population. Case reports are described in children with risk factors such as immunocompromised. Outbreaks are commonly associated with buildings or structures that have complex water systems like hospitals. The most likely sources of infection include water used for showering, cooling towers, respiratory therapy equipment, humidifiers and other health care devices. Clinical presentation does not distinguish from other respiratory infections, difficult access to rapid diagnostic methods and empirical antibiotic treatment not effective are all factors that delay diagnosis and subsequent control measures, leading to an increased spread risk. Methods A case of Legionnaires' disease was rapidly detected in an immunocompromised pediatric patient with nosocomial pneumonia using BIOFIRE® FILMARRAY® Pneumonia Plus Panel and urinary antigen. The Infection Control Committee was reported, initiating control and surveillance measures: hospital's water sources were studied and patients with nosocomial pneumonia with no etiological diagnosis were studied in the same way. Results L. pneumophila was isolated in a water tank's sample with the same restriction profile as the clinical sample. A water safety plan was implemented: the water tank's temperature was raised to 65°C and hyperchlorinated to maintain an adequate level of disinfectant. Control cultures were performed, all negative. No new cases were detected. Conclusions It is important to consider Legionella pneumophila as a potential agent of pneumonia in children since it can result in life-threatening health conditions and severe nosocomial outbreak infection. The incorporation of rapid molecular or antigen diagnostic techniques allow prompt diagnosis and treatment, as well as rapid environmental and clinical surveillance measures that can reduce morbidity and mortality. When detecting Legionella infection, it is essential to identify the environmental focus so as to prevent the development of new cases.

Characteristics of the Mycoplasma pneumoniae Epidemic from 2019 to 2020 in Korea: Macrolide Resistance and Co-Infection Trends.

Mycoplasma pneumoniae, a major etiological agent of community-acquired pneumonia, exhibits distinct cyclic epidemic patterns recurring every three to five years. Several cases of co-infection with severe acute respiratory syndrome coronavirus 2 have been reported globally, resulting in unfavorable clinical manifestations. This study investigated the epidemiological features of the recent M. pneumoniae outbreak (May 2019-April 2020) using retrospective data from the last five years. Molecular test data for macrolide resistance and co-infection were obtained from the Seegene Medical Foundation. National medical expenditure and hospitalization rates were analyzed using data from The Health Insurance Review and Assessment Service of Korea. The macrolide resistance rate was 69.67%, peaking at 71.30% during the epidemic period, which was considerably higher than the 60.89% rate during non-epidemic periods. The co-infection rate with other respiratory pathogens was 88.49%; macrolide-resistant M. pneumoniae strains showed a 2.33% higher co-infection rate than the susceptible strains. The epidemic period had 15.43% higher hospitalization and 78.27% higher medical budget expenditure per patient than non-epidemic periods. The increased rates of macrolide resistance and co-infection observed in macrolide-resistant M. pneumoniae during the epidemic period highlight the importance of monitoring future outbreaks, especially considering macrolide resistance and the risk of co-infection with other pathogens.

Synthesis of Fluorescent-Labeled Nucleotides for Labeling of Isothermal Amplification Products

The synthesis of fluorescently labeled nucleotides containing a zwitterionic indodicarbocyanine dye attached via a trans-alkene spacer at the C5 position of the pyrimidine base was carried out, and their substrate efficiency was tested under conditions of recombinase polymerase amplification (RPA). As a result of RPA, the formation of full-sized target products of the ebpS gene fragment of the causative agent of bacterial pneumonia (Staphylococcus aureus) and a high density of fluorescent label embedding occurred.

Identification of repurposable drug targets in Mycoplasma pneumoniae using subtractive genomics, molecular docking and dynamics simulation

Mycoplasma pneumoniae is a significant causative agent of community-acquired pneumonia, causing acute inflammation in the upper and lower respiratory tract as well as extrapulmonary syndromes. In particular, the elderly and infants are at greater risk of developing severe, life-threatening pneumonia caused by M. pneumoniae. Yet, the global increase in antimicrobial resistance against antibiotics for the treatment of M. pneumoniae infection highlights the urgent need to explore novel drug targets. To this end, bioinformatics approaches, such as subtractive genomics, can be employed to identify specific metabolic pathways and essential proteins unique to the pathogen that could be potential targets for new drugs. In this study, we implemented a subtractive genomics approach to identify 61 metabolic pathways and 42 essential proteins that are unique to M. pneumoniae. A subsequent screening in the DrugBank database revealed three druggable proteins with similarity to FDA-approved small-molecule drugs, and finally, the compound CHEBI:97093 was identified as a promising novel putative drug target. These findings can provide crucial insights for the development of highly effective drugs that selectively inhibit the pathogen-specific metabolic pathways, leading to better management and treatment of M. pneumoniae infections.

Value of pneumococcal PCR in respiratory samples for exclusion of pneumococcal pneumonia.

Streptococcus pneumoniae is the main aetiological agent in bacterial pneumonia. Therefore pneumococcal PCR is often included in respiratory multiplex PCR panels, both commercial and in-house. But respiratory PCR results for S. pneumoniae are difficult to interpret due to frequent non-pathogenic colonization on the mucosal surface of the upper airways with pneumococci or to cross-reaction of the PCR target in non-pneumococcal streptococci. In this study we investigated the value of lytA gene pneumococcal PCR in patients presenting with pneumonia. To assess the utility of lytA gene detection for S. pneumoniae in a respiratory multiplex quantitative PCR (qPCR) panel for patients presenting with pneumonia. A retrospective study was conducted for lytA gene results as target for S. pneumoniae in hospitalized patients who were diagnosed with pneumonia and for which a respiratory multiplex PCR panel was performed. Patients were classified as 'probable', 'possible' or 'unlikely' of having a pneumococcal pneumonia. A sensitivity of 71.4% and specificity of 89.6% were found, corresponding to a negative predictive value and positive predictive value of 97.6% and 34.2%, respectively, when considering 'probable' versus 'possible/unlikely'. In the PCR-positive cases we found a statistically significant difference in semi-quantitative Ct values between the 'probable' and the 'possible/unlikely' groups. We conclude that a negative qPCR for the lytA gene in a respiratory sample is highly predictive of a negative S. pneumoniae culture and is possibly sufficient to exclude S. pneumoniae as a causative agent. Respiratory pneumococcal PCR has a high negative predictive value for pneumococcal disease but the positive predictive value is low.

Features of clinical manifestations and diagnosis of comorbidity of disseminated pulmonary tuberculosis, coronavirus, pneumocystis and pneumococcal pneumonia in patients with late stages of HIV infection with immunodeficiency

Aim. To study the features of clinical manifestations and diagnosis of comorbidity of disseminated pulmonary tuberculosis, coronavirus, pneumocystis and pneumococcal pneumonia in patients with late stages of HIV infection with immunodeficiency.
 Materials and methods. The prospective study included 120 newly identified patients with disseminated pulmonary tuberculosis with Mycobacterium tuberculosis, stage IVB of HIV infection, in the phase of progression and in the absence of antiretroviral therapy, aged 2953 years, who were randomized into 1A and 2A main groups and 1B and 2B comparison groups. Group 1A included 29 patients with comorbidity and pneumocystis pneumonia and group 2A 31 patients with comorbidity of disseminated pulmonary tuberculosis, coronovirus pneumococcal pneumonia, and group 1B and 2B comprised 29 and 31 similar patients, but without coronovirus pneumonia. To diagnose coronavirus pneumonia, PCR of SARS-CoV-2 RNA was used in smears from the nasopharynx and oropharynx, in sputum or in endotracheal aspirate. To detect Pneumocystis jirovecii, the causative agent of pneumocystis pneumonia, a microscopic examination of diagnostic material from the respiratory tract with RomanovskyGiemse and GrokottGmri coloration was carried out, and to detect Streptococcus pneumoniae, the causative agent of pneumococcal pneumonia, the diagnostic material was seeded on special nutrient media with determination of the drug resistance of the resulting culture to broad-spectrum antibiotics. Statistical data processing was carried out using the Microsoft Office Excel 2019 program with the calculation of the average in the group and the standard error of the average, confidence interval.
 Results. The comorbidity of disseminated pulmonary tuberculosis, coronavirus, pneumocystis and pneumococcal pneumonia in patients in the late stages of HIV infection, in the phase of progression and in the absence of antiretroviral therapy was characterized by severe immunodeficiency, generalization of tuberculosis with multiple extrapulmonary lesions and severe pneumonia. This determines the similarity of clinical manifestations and respiratory symptoms, and also makes it difficult to visualize computed tomographic changes consisting of a complex simultaneous combination of four pathological syndromes: dissemination, pleural pathology, increased pulmonary pattern and adenopathy. Simultaneous layering of several pathologies with the same type of clinical manifestations and computed tomographic changes requires a comprehensive etiological diagnosis of specific diseases to prescribe timely comprehensive treatment and reduce the lethality of this heavy contingent of patients.
 Conclusion. Patients with disseminated pulmonary tuberculosis and HIV infection who are registered in the office of tuberculosis care for HIV-infected in the tuberculosis dispensary represent a high risk group of COVID-19 infection and the development of coronavirus pneumonia, and with severe immunodeficiency, pneumocystis and pneumococcal pneumonia, should be regularly subjected to preventive studies for timely detection of COVID-19, coronavirus, pneumocystis and pneumococcal pneumonia for the purpose of their emergency isolation and timely treatment.

Comparative Analysis of the Pathogen Structure in Patients with Community-Acquired and Nosocomial Pneumonia in Medical Organizations of the Rostov, Tyumen Regions and Khabarovsk Territory at the Current Stage of a New Coronavirus Infection Pandemic

The aim of the study was to conduct a comparative analysis of the spectrum and antibiotic resistance of secondary pneumonia pathogens isolated in the territories of the Rostov, Tyumen Regions and Khabarovsk Territory against the background of a new coronavirus infection pandemic.Materials and methods. We investigated sputum samples from coronavirus-positive and coronavirus-negative patients with community-acquired pneumonia from medical organizations using bacteriological method, PCR mass spectrometry.Results and discussion. The study of the etiological structure of secondary pneumonia agents isolated from patients in medical organizations of the Southern, Ural and Far Eastern Federal Districts has revealed that the dominant cultures in SARS‑CoV‑2 “+” and SARS‑CoV‑2 “–” patients were yeast and yeast-like fungi. It has been found that under diversity of isolated fungi, Candida albicans species prevailed. The bacterial microflora is represented by a variety of gram-positive and gram-negative bacteria, of which Staphylococcus aureus and Klebsiella pneumoniaе were most often present in sputum. It has also been established that even before hospitalization of patients, community-acquired pneumonia could be caused by microorganisms of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniaе, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.), which are usually considered as polyantibiotic-resistant pathogens of nosocomial infections. Moreover, in coronavirus-positive patients with secondary community-acquired pneumonia, those pathogens were isolated 2–3 times more frequently than in coronavirus-negative ones. Assessment of sensitivity/resistance of isolated strains to antibacterial drugs has revealed a general trend: the majority of the strains, regardless of the type, were characterized by a narrow spectrum of sensitivity, having 3 or more markers of antibiotic resistance. This confirms the necessity and expediency of microbiological support of the patient during the entire infectious process. The most adequate drugs of choice, providing activity against 60–70 % of strains of the Enterobacteriaceae family, are amikacin and cefoperazone/sulbactam.

Гостра деструктивна пневмонія та піофібриноторакс в експерименті

Nowadays, the problem of diagnosis and treatment of purulent-destructive diseases of the bronchopulmonary system in children remains relevant, that is connected with a large number of pulmonary-pleural forms and complications of acute necrotizing pneumonia. Purpose - to establish the correlation of changes in lung tissue and the pleural cavity of a growing organism, depending on the causative agents of pneumonia, elucidation and study of the mechanisms of pyofibrothorax formation and development. Materials and methods. The characteristic features of histopathological tissue changes in the lung parenchyma and adjacent tissues have been analyzed (visceral and parietal pleura, adhesion formation sections) of 45 immature laboratory rats, divided into 5 groups: the control group (intact animals), the Group 2 - contaminated with Klebsiella pneumoniae, the Group 3 - Staphylococcus aureus, the Group 4 - Candida albicans and the Group 5 with mixed flora (Klebsiella pneumoniae+Staphylococcus aureus) adding fungal infection (Candida albicans). Results. It was proved that the formation of a massive pyofibrinothorax is enhanced by mixed flora with the addition of fungi, that is more pronounced in the fifth series (group) of the experiment. Taking into account the data obtained experimentally, we consider whether fungi are Lukianenkoobtained in cultures of patients’ flora, they should be classified as a risk group of fibrothorax formation. Conclusions. Morphological changes of tissues under the influence of the infectious agent reached their maximum on the ninth day of the experiment (p<0.05). The pattern of necrotizing pneumonia is more pronounced in the Group 5 of experimental animals, which morphologically manifests itself in a more massive inflammatory infiltration (mostly due to rod- and segmented-nuclear neutrophils), foci of destruction and abscessation of the lung parenchyma and has a direct association with the polymicrobial etiology of the disease (Klebsiella pneumoniae+Staphylococcus /taureus+Candida albicans) (p<0.05). The formation of a massive pyofibrinothorax is enhanced by mixed flora, especially with the addition of fungi. The study was carried out in accordance with the principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, as well as the law of Ukraine. The research protocol was approved by the Local Ethics Committee of the institution mentioned in the article. No conflict of interests was declared by the authors.

De novo genome assembly resolving repetitive structures enables genomic analysis of 35 European Mycoplasmopsis bovis strains

Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.

Assessing the utilization of antimicrobial agents in pediatric pneumonia during the era of the 13-valent pneumococcal conjugate vaccine: A retrospective, single-center study

Background and purposePneumonia and bronchopneumonia are the most common infectious diseases in children. This study aimed to analyze changes in causative pathogens and antibiotic use for bronchopneumonia or pneumonia after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in children. MethodsThis retrospective study was conducted from 2009 to 2019. Hospitalized children aged 6 months–3 years with a discharge diagnosis of bronchopneumonia or pneumonia were included to analyze changes in the potential mismatch between the diagnosed pathogen and antibiotic use. ResultsThe cohort comprised 1100 patients, including 648 (59%) and 452 (41%) with a discharge diagnosis of bronchopneumonia and pneumonia, respectively. The trend of viral pneumonia increased every year (rs = 0.101, p < 0.05) Antibiotics were administered in 97% patients, with an increasing annual trend in macrolide use (rs = 0.031, p = 0.009). Regarding antibiotic utilization, no significant variations were observed in the days of therapy (DOT) (rs = 0.076, p = 0.208) or length of therapy (LOT) (rs = −0.027, p = 0.534) per patient-year throughout the study duration. Interestingly, the LOT for combined therapy with macrolides and first-line beta-lactams was high (rs = 0.333, p = 0.028). In viral pneumonia treatment, neither the DOT nor LOT exhibited significant variations (rs = −0.006, p = 0.787 and rs = −0.156, p = 0.398). ConclusionAfter the introduction of PCV13 in Taiwan, no decrease in antibiotic use has been observed among children aged 6 months–3 years with a discharge diagnosis of bronchopneumonia and pneumonia.

Comparative study of the etiology of nosocomial bacteremic pneumonia in ventilated and non-ventilated patients: a 10-year experience in an institution.

The etiology of nosocomial pneumonia (NP) in non-mechanically ventilated patients [hospital-acquired pneumonia (HAP)] is poorly understood because of difficulties in obtaining reliable respiratory samples. While it is thought to resemble that of mechanically ventilated patients [ventilator-associated pneumonia (VAP)], few studies compare etiology in both groups. We compared the etiology of bacteremic NP (bNP) episodes in HAP and VAP over 10 years in our institution. We defined NP in adults according to the American Thoracic Society criteria. bNP episodes were defined as significant isolates in ≥1 blood culture concordant with ≥1 respiratory pathogen isolated with <7 days of difference. During 2010-2019, 188 patients were included. The comparison between HAP (n = 104) and VAP (n = 84) revealed the following factors: male sex (80.8%/63.1%; P < 0.01); median age (69.3/67.8 y-o; P = 0.602); etiology [Staphylococcus aureus (40.49%/21.4%; P < 0.01), Enterobacterales (35.6%/39.3%; P = 0.601), and Pseudomonas aeruginosa (14.4%/34.5%; P < 0.01)]. Microorganisms were considered multidrug-resistant in 29.8%/21.4% of cases, respectively (P = 0.193). Median hospital stay in HAP/VAP was 45.0/53.5 days (P = 0.255), mortality was 55.8%/53.6% (P = 0.770), and related mortality was 45.2%/35.7% (P = 0.233). The etiology of bNP in hospitalized patients is similar but not identical in HAP and VAP. The differences included a higher prevalence of S. aureus in HAP and Pseudomonas aeruginosa in VAP. bNP is a serious disease, with mortality >40%.ImportanceThis study on bacteremic nosocomial pneumonia (bNP) demonstrates the importance of this condition both in patients undergoing and not undergoing mechanical ventilation. Staphylococcus aureus, Enterobacterales, and non-fermenting Gram-negative bacilli are all causative agents in ventilator-associated pneumonia (VAP) and hospital-acquired pneumonia (HAP), with a predominance of S. aureus in HAP and of Pseudomonas aeruginosa in VAP. Mortality in this condition is very high. Therefore, new therapeutic and preventive approaches should be sought.

Aetiological agents of adult community-acquired pneumonia in Japan: systematic review and meta-analysis of published data

ObjectiveEpidemiological information is essential in providing appropriate empiric antimicrobial therapy for pneumonia. This study aimed to clarify the epidemiology of community-acquired pneumonia (CAP) by conducting a systematic review of published...

A Recombinant Chimera Vaccine Composed of LTB and Mycoplasma hyopneumoniae Antigens P97R1, mhp390 and P46 Elicits Cellular Immunologic Response in Mice.

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia.

Evaluation of the Effectiveness and Use of Anti-Methicillin-Resistant Staphylococcus aureus Agents for Aspiration Pneumonia in Older Patients Using a Nationwide Japanese Administrative Database.

Studies indicated potential harm from empirical broad-spectrum therapy. A recent study of hospitalizations for community-acquired pneumonia suggested that empirical anti-methicillin-resistant Staphylococcus aureus (MRSA) therapy was associated with an increased risk of death and other complications. However, limited evidence supports empirical anti-MRSA therapy for older patients with aspiration pneumonia. In a nationwide Japanese database, patients aged ≥65 years on admission with aspiration pneumonia were analyzed. Patients were divided based on presence of respiratory failure and further sub-categorized based on their condition within 3 days of hospital admission, either receiving a combination of anti-MRSA agents and other antibiotics, or not using MRSA agents. An inverse probability weighting method with estimated propensity scores was used. Out of 81,306 eligible patients, 55,098 had respiratory failure, and 26,208 did not. In the group with and without respiratory failure, 0.93% and 0.42% of the patients, respectively, received anti-MRSA agents. In patients with respiratory failure, in-hospital mortality (31.38% vs. 19.03%, p < 0.001), 30-day mortality, and 90-day mortality were significantly higher, and oxygen administration length was significantly longer in the anti-MRSA agent combination group. Anti-MRSA agent combination use did not improve the outcomes in older patients with aspiration pneumonia and respiratory failure, and should be carefully and comprehensively considered.

Insight into the Mechanism of Interactions between the LL-37 Peptide and Model Membranes of Legionella gormanii Bacteria.

Legionella gormanii is a fastidious, Gram-negative bacterium known to be the etiological agent of atypical community-acquired pneumonia. The human cathelicidin LL-37 exhibits a dose-dependent bactericidal effect on L. gormanii. The LL-37 peptide at the concentration of 10 µM causes the bacteria to become viable but not cultured. The antibacterial activity of the peptide is attributed to its effective binding to the bacterial membrane, as demonstrated by the fluorescence lifetime imaging microscopy. In this study, to mimic the L. gormanii membranes and their response to the antimicrobial peptide, Langmuir monolayers were used with the addition of the LL-37 peptide to the subphase of the Langmuir trough to represent the extracellular fluid. The properties of the model membranes (Langmuir monolayers) formed by phospholipids (PL) isolated from the L. gormanii bacteria cultured on the non-supplemented (PL-choline) and choline-supplemented (PL+choline) medium were determined, along with the effect of the LL-37 peptide on the intermolecular interactions, packing, and ordering under the monolayer compression. Penetration tests at the constant surface pressure were carried out to investigate the mechanism of the LL-37 peptide action on the model membranes. The peptide binds to the anionic bacterial membranes preferentially, due to its positive charge. Upon binding, the LL-37 peptide can penetrate into the hydrophobic tails of phospholipids, destabilizing membrane integrity. The above process can entail membrane disruption and ultimately cell death. The ability to evoke such a great membrane destabilization is dependent on the share of electrostatic, hydrogen bonding and Lifshitz-van der Waals LL-37-PL interactions. Thus, the LL-37 peptide action depends on the changes in the lipid membrane composition caused by the utilization of exogenous choline by the L. gormanii.

The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires' disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

P02 Antimicrobial susceptibility of microorganisms isolated in community-acquired pneumonia in drug-dependent patients

Abstract Objectives To investigate the nature and susceptibility of community-acquired pneumonia (CAP) pathogens in drug-dependent patients. Materials and methods The susceptibility of community-acquired pneumonia pathogens isolated from sputum was studied in 31 drug-dependent patients. 13 (42%) were women and 18 (58%) were men. The average age of this group was 28.6 ± 1.2 years. The antimicrobial activity of the main groups of antibiotics was studied in comparison with the susceptibility of the archive Staphylococcus aureus ATCC25923 strain. Results In total, 51 strains of microorganisms were isolated, among which S. aureus (19 strains, 37.3%) and fungi of the genus Candida (16 strains, 31.3%) prevailed, while in 15 cases, bacteria were inoculated in the form of monoculture, and in 16 cases in the form of associations of bacteria and fungi of the genus Candida. When studying the susceptibility of the isolated strains of microorganisms to the main groups of antibiotics, their high (P ≤ 0.05) resistance to the main groups of antimicrobial drugs used in standard treatment regimens for CAP was established. Conclusions CAP in drug-dependent patients has a severe complicated course due to the combination of highly virulent bacterial and fungal infections. The causative agents of community-acquired pneumonia in drug-dependent patients have low susceptibility to the main groups of antibiotics used in the treatment of this pathology.

P18 Value of pneumococcal PCR in respiratory samples for exclusion of pneumococcal pneumonia

Abstract Background Streptococcus pneumoniae is the main aetiological agent in bacterial pneumonia, making it an essential component of respiratory multiplex PCR panels. Despite its relevance, PCR test results are difficult to interpret due to frequent non-pathogenic colonization on the mucosal surface of the upper airways. Methods A retrospective study was conducted for lytA-gene results as target for S. pneumoniae in hospitalized patients that were diagnosed with pneumonia and on which a respiratory multiplex PCR panel was performed. Patients were classified as probable, possible or unlikely of having a pneumococcal pneumonia. Results No difference in semi-quantitative Ct-values could be observed between the different groups. Sensitivity of 71.4% and specificity of 89.6% was found, corresponding with a negative predictive value and positive predictive value of 97.6% and 34.2%, respectively, when considering probable versus possible/unlikely. Conclusions We conclude that a negative qPCR for the lytA-gene is a good marker for the inability to culture S. pneumoniae in a respiratory sample and is possibly sufficient to exclude S. pneumoniae as a causative agent, due to which we can regard it as a relevant addition to a syndromic multiplex PCR panel.

Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.