Nasopharyngeal Microflora Research Articles

Multi-omics investigation into long-distance road transportation effects on respiratory health and immunometabolic responses in calves.

Long-distance road transportation is a common practice in the beef industry, frequently resulting in bovine respiratory disease (BRD) and compromised growth performance. However, a comprehensive investigation integrating clinical performance, physiological conditions, and nasopharyngeal microflora remains lacking. This study aimed to evaluate the respiratory health and immunometabolic status of 54 beef calves subjected to a 3000-km journey. The respiratory health of calves was monitored over 60 days post-arrival using a modified clinical scoring system. Nasopharyngeal microflora and venous blood samples were collected at 3 time points: before transportation (A), 30 days post-arrival (B), and 60 days post-arrival (C), for 16S rRNA microbiomics, whole-blood transcriptomics, serum metabolomics, and laboratory assays. Within the first week post-arrival, the appetite and mental scores of calves dropped to zero, while other respiratory-related scores progressively declined over the 60 days. The α-diversity of nasopharyngeal microflora in calves was similar at time points A and B, both significantly higher than at time point C. The structure of these microbial communities varied significantly across different time points, with a notably higher relative abundance of BRD-related genera, such as Pasteurella and Mannheimia, detected at time point A compared to B and C. The composition and gene expression profiles of circulating blood cells at time point A were significantly different from those at B and C. Specifically, higher expression levels of oxidative- and inflammatory-related genes, cytokines, and enzymes were observed at time point A compared to B and C. Higher levels of catabolism-related metabolites and enzymes were detected at time point A, while higher levels of anabolism-related metabolites and enzymes were observed at time points B and C. Additionally, significant correlations were found among microorganisms, genes, and metabolites with differing abundances, expression levels, and concentrations across time points. Stronger correlations were observed between calves' performance and nasopharyngeal microflora and immunometabolic status at time point A compared to B or C. Collectively, these results confirm that 3000 km of road transportation significantly alters the composition and gene expression profiles of circulating white blood cells in calves, affects their metabolic processes, disrupts the balance of the respiratory microbial community, and leads to pronounced respiratory symptoms that persist for at least 60 days. During this period, the influenced composition and gene expression of circulating blood cells, metabolic processes, and nasopharyngeal microbial community gradually return to equilibrium, and the respiratory symptoms gradually diminish. This observational research indicates that transportation induces BRD in calves by disrupting the homeostasis of their immune function, metabolic processes, and nasopharyngeal microbial community. However, these results and their underlying molecular mechanisms warrant further validation through well-designed in vivo and in vitro confirmatory experiments with larger sample size. Video Abstract.

Multi-omics reveals that alkaline mineral water improves the respiratory health and growth performance of transported calves

BackgroundLong-distance transportation, a frequent practice in the cattle industry, stresses calves and results in morbidity, mortality, and growth suppression, leading to welfare concerns and economic losses. Alkaline mineral water (AMW) is an electrolyte additive containing multiple mineral elements and shows stress-mitigating effects on humans and bovines.ResultsHere, we monitored the respiratory health status and growth performance of 60 Simmental calves subjected to 30 hours of road transportation using a clinical scoring system. Within the three days of commingling before the transportation and 30 days after the transportation, calves in the AMW group (n = 30) were supplied with AMW, while calves in the Control group (n = 29) were not. On three specific days, namely the day before transportation (day -3), the 30th day (day 30), and the 60th day (day 60) after transportation, sets of venous blood, serum, and nasopharyngeal swab samples were collected from 20 calves (10 from each group) for routine blood testing, whole blood transcriptomic sequencing, serology detection, serum untargeted metabolic sequencing, and 16S rRNA gene sequencing. The field data showed that calves in the AMW group displayed lower rectal temperatures (38.967 ℃ vs. 39.022 ℃; p = 0.004), respiratory scores (0.079 vs. 0.144; p < 0.001), appetite scores (0.024 vs. 0.055; p < 0.001), ocular and ear scores (0.185 vs. 0.338; p < 0.001), nasal discharge scores (0.143 vs. 0.241; p < 0.001), and higher body weight gains (30.870 kg vs. 7.552 kg; p < 0.001). The outcomes of laboratory and high throughput sequencing data revealed that the calves in the AMW group demonstrated higher cellular and humoral immunities, antioxidant capacities, lower inflammatory levels, and intestinal absorption and lipogenesis on days -3 and 60. The nasopharynx 16S rRNA gene microbiome analysis revealed the different composition and structure of the nasopharyngeal microflora in the two groups of calves on day 30. Joint analysis of multi-omics revealed that on days -3 and 30, bile secretion was a shared pathway enriched by differentially expressed genes and metabolites, and there were strong correlations between the differentially expressed metabolites and the main genera in the nasopharynx.ConclusionsThese results suggest that AMW supplementation enhances peripheral immunity, nutrition absorption, and metabolic processes, subsequently affecting the nasopharyngeal microbiota and improving the respiratory health and growth performance of transported calves. This investigation provided a practical approach to mitigate transportation stress and explored its underlying mechanisms, which are beneficial for the development of the livestock industry.9KT6u9sWWs8ZH5U7yZx1qCVideo

Some aspects of nasopharyngeal microflora in children with psoriasis

Objective — to study the state of nasopharyngeal microflora in children with psoriasis depending on the clinical features of dermatosis and determine antibiotic susceptibility of isolated microorganisms. Materials and methods. The isolation of nasopharyngeal microorganisms and determination of their antibiotic susceptibility were performed in 57 children with psoriasis aged 4 to 17 using the bacteriological method (culture). Microbial growth was assessed according to four degrees of growth. Susceptibility to antibacterial drugs was evaluated based on the following criteria: sensitive, moderately resistant, and resistant. Study materials were statistically processed using the StatTech v. 3.1 software. Results and discussion. Most often, Staphylococcus aureus (S. aureus) associations were cultured from the nasopharynx (56.15 %) of children with psoriasis — in 12.28 % of cases, Enterococcus faecium (Ent. faecium) — in 21.05 %. Statistically significant differences in children with psoriasis were found between the colony growth rates of Ent. faecium and S. aureus (p &lt; 0.001): massive microbial growth of Ent. faecium prevailed (64.91 %), and S. aureus was not isolated at all in the majority of children (29.82 %). Ent. faecium growth rates were statistically significantly different (p = 0.016) depending on psoriasis severity according to PASI: massive growth of microorganisms at PASI &gt; 10 was found in 87.5 % of cases, and at PASI ≤ 10 — in 54.2 % of cases. The proportion of Ent. faecium resistant to antimicrobial drugs (43.18 ± 7.47) % was found to be more than two times higher than that of S. aureus (20.00 ± 6.32) %. The susceptibility testing for each microorganism individually showed that enterococci were 28 to 100 % susceptible to antibacterial drugs, while staphylococci had 42 to 100 % susceptibility. Statistically significant differences were found between the percentage of sensitivity of Ent. faecium to antibacterial drugs between the group of children with the first diagnosis of psoriasis and the group of children with its recurrences (p = 0.005), as well as by the duration of the disease (p &lt; 0.001). Conclusions. The results of the study of children with psoriasis showed that there were bacterial associations isolated from the nasopharynx in the most significant number of cases with the predominance of Ent. faecium, which had massive growth in almost 65 % of cases and affected psoriasis severity according to PASI (OR 5.923; 95 % CI 1.387—25.300). The proportion of Ent. faecium resistant to antibiotics was higher than of S. aureus (p &lt; 0.05), but with increasing duration of psoriasis, Ent. faecium susceptibility to antimicrobials increased (r = 0.439; p = 0.004). In general, S. aureus isolated from the nasopharynx of children with psoriasis had more pronounced antibiotic susceptibility than Ent. faecium.

The state of the nasopharyngeal microbiome in healthy people and in patients with chronic nasopharyngitis

Topicality: The chronic nasopharyngitis (CNP) morbidity rate tends to increase in childhood and adenoid removal is one of the most common of all surgeries in children. The state of the nasopharyngeal microbiome plays the significant role in the development of inflammatory processes of nasopharynx and adenoid hypertrophy and it is considered that the great progress can be made through its correction in conservative treatment and prevention of recurrence after surgery. Traditional cultural studies which reflect only a small proportion of bacteria present in the nasopharynx cannot describe the real state of microbiome. Therefore, the study of its quantitative and qualitative composit ion is essent ial for justified treatment. Aim: to examine the state of the nasopharyngeal microbiome in patients with CNP associated with EpsteinBarr virus and compare its characteristics with the severity of disease. Material and methods: 20 healthy persons and 32 patients diagnosed with CNP associated with EpsteinBarr virus have been included in the study. The qualitative composition of nasopharyngeal microflora and its quantitative characteristics have been studied by determining t itres of colony-forming units (CFU). The findings have been compared with the features of the clinical course of disease. Results: The healthy individuals of the control group have Lactobacillus spp. and Bifidobacterium spp., aerobic S. salivarius, which belong to commensal microorganisms, as the basis of the nasopharyngeal microbiome. There has been seen the preservation of the population level of commensal flora: lactobacilli, bifidobacteria, salivary streptococci and appearance of opportunistic flora (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli) in small titres in the patients with mild CNP. During the moderate/severe course, there has been observed a sharp decline in the population level of commensal flora and an increase in the titer of opportunistic flora to the population level of commensals. In severe cases, the preservation of high titers of opportunistic flora, complete absence of commensal one and emergence of pathogenic microflora (Pneumococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa and yeast-like fungi of the Candida type) can be seen. All patients have been observed the presence of microbiome disorders, regardless of its quantitative and qualitative changes, which are accompanied by hypertrophy of the pharyngeal tonsil. Conclusions: Dysfunction of the microbiome, i.e. changes in the quantitative and qualitative composition of microorganisms in patients with CNP, is important in the course of disease. Minor changes that are determined by a reduced level of commensal flora are associated with the mild disease course. If there is a significant reduction in the levels of commensals and emergence of opportunistic flora, the moderate/severe disease course can be observed. The complete absence of commensal flora, high titers of opportunistic pathogens and emergence of pathogenic flora including Candida albicans are characterized by a severe disease course.

RESPIRATORY MICROBIOME AND ITS RELATIONSHIP WITH INFLAMMATORY MARKERS.

The aim: This study aims to investigate and analyze the microbiome of the nasopharyngeal zone in acute respiratory infections (ARI) and their relationship with inflammatory markers. Materials and methods: Examination of 112 children (10-14 years old) with acute respiratory infections (ARI) of the upper respiratory tract was carried out. The control group consisted of 25 healthy children identical in age and examination parameters. Results: When analyzing the microflora of the nasopharynx of patients, 29,0% of strains were gram-positive bacteria and 71,0% were gram-negative bacteria (Escherichia coli representing 37,0%). Biochemical examination of the biomaterial revealed the presence of sucrase (n=69), maltase (n=87), lactorepoxidase (n-89) and alcohols - sorbitol (hexahydric alcohol, n=102), mannitol (hexahydric alcohol, n=84), xylitol (pentahydric alcohol, n=86). Regarding the markers of inflammatory response, the following dynamics was noted: increase in the level of IgM (3,91 ± 1,79 g/l, p<0,01) by 2,2 times, elevation of Ig G level by 10 times (145, 91 ± 53,04 g/l, p< 0,01), slightly higher than the reference values IgE level. In addition, increased IL-1, IL-4, IL-6, γ-IFN, TNF-α, Neopterin levels were detected. The level of Thyroid stimulating hormone (TSH) was significantly different compared to the control group (0,62 ± 0,57 vs. 1,98 ± 0,30 mIU/ ml, p< 0,01), but within the reference values. Conclusions: Predominance of Gram-negative bacteria in the nasopharyngeal microflora of patients along with elevated inflammatory markers and lactop-eroxydase enzyme predominance was detected in the study.

A Systematic Review of the Literature Examining the Effects of Cigarette Smoke and e-Cigarette Vapor on the Virulence of Human Pathogenic Bacteria.

The bioactive chemicals in cigarette smoke (CS) and e-cigarette vapor (EV) may affect pathogenic bacteria in the nasopharyngeal microflora, which may have implications on the pathophysiology of respiratory infections in cigarette smokers and e-cigarette users. In this systematic review, we seek to synthesize the research evidence supporting this hypothesis. To address the central research question, “what is known from the published, peer-reviewed literature about the effects of cigarette smoke or e-cigarette vapor exposure on the physiology of human pathogenic bacteria?”, we screened the PubMed®, Web of ScienceTM, and ScienceDirect databases for reports examining the virulence characteristics and gene expression in human pathogenic bacteria exposed to either CS or EV. The principal conclusion from our analysis is that exposure to either CS or EV induces the virulence of respiratory pathogenic bacteria in a strain-dependent manner, which may in turn facilitate respiratory infections in cigarette smokers and e-cigarette users. In addition, we present evidence that nicotine and reactive oxygen species are the main chemicals responsible for CS/EV-mediated alterations in bacterial physiology. We note limitations that this review does not examine reports describing the alterations in host respiratory physiology or nasopharyngeal dysbiosis caused by CS/EV exposure. Future research to determine whether CS/EV-mediated augmentation of bacterial virulence indeed plays a role in human respiratory tract infections is warranted.

Evaluation of the two in-patient hospitals on potential environmental hazard during the period of new coronavirus infection in the Khabarovsk city (december 2020 — march 2021)

Microbiological monitoring after infectious diseases in the system of epidemiological surveillance implies simultaneous pathogen identification both among patients and in hospital environment. Our aim is to assess potential hospital environmental hazard for the two in-patient infectious disease hospitals of the Khabarovsk city by using bacteriological and epidemiological analysis during new coronavirus disease pandemic. Materials and methods. Bacteriological assessment of nasopharyngeal microflora in 241 patients suffering from community-acquired pneumonia that were hospitalized in the two prevention and treatment facilities of the Khabarovsk city was performed. Sanitary-bacteriological control of hospital environment (428 hospital environment samples and 91 air samples) was carried out in parallel. Bacteriological assessment was performed with classical methods. Identification of isolated bacteriological pathogens and evaluation of drug-resistant strains were carried out by utilizing bacteriological analyzer Vitek 2 Compact. Results. Nine different pathogens (Pseudomonas aeruginosa, Pseudomonas stutzeri, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Pantoea, Enterococcus faecium, Staphylococcus haemolyticus) were isolated in 20 out of 428 samples 4.7% [2.76.7]. Half of isolated agents 2.3% [0.93.8] were represented by drug-resistant isolates (10 out of 20 isolates) including 5 carbapenem-resistant isolates (Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae) and 5 isolates with multiple drug resistance (Enterobacter cloacae, Pantoea, Enterococcus faecium, Staphylococcus haemolyticus). Air samples contained pathogenic biological agents found in 6 out of 91 samples 6.6% [1.511.7], and half of them 3.3% [0.67.9] were identified as drug-resistant variants, including S. aureus и S. haemolyticus. One of the surveyed hospitals was recognized as more hazardous due to microflora isolated from intensive care unit (A. baumannii and P. aeruginosa were resistant to 3rd4th generation cephalosporins and carbapenems). Conclusion. Revealed circulation of wide range of microorganisms isolated from environment of two in-patient hospitals indicates high risk of healthcare-associated infections formation. Intensive care units can serve as a reservoir of healthcare-associated infections due to high percentage of patients with severe disease cases (main reservoir of drug-resistant strains).

RNA thermosensors facilitate Streptococcus pneumoniae and Haemophilus influenzae immune evasion.

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted respiratory pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases, remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signals to pathogens. Here, we investigate whether these respiratory pathogens can sense environmental temperature to evade host complement-mediated killing. We show that productions of two vital virulence factors and vaccine components, the polysaccharide capsules and factor H binding proteins, are temperature dependent, thus influencing serum/opsonophagocytic killing of the bacteria. We identify and characterise four novel RNA thermosensors in S. pneumoniae and H. influenzae, responsible for capsular biosynthesis and production of factor H binding proteins. Our data suggest that these bacteria might have independently co-evolved thermosensing abilities with different RNA sequences but distinct secondary structures to evade the immune system.

Capacity To Utilize Raffinose Dictates Pneumococcal Disease Phenotype.

Streptococcus pneumoniae is commonly carried asymptomatically in the human nasopharynx, but it also causes serious and invasive diseases such as pneumonia, bacteremia, and meningitis, as well as less serious but highly prevalent infections such as otitis media. We have previously shown that closely related pneumococci (of the same capsular serotype and multilocus sequence type [ST]) can display distinct pathogenic profiles in mice that correlate with clinical isolation site (e.g., blood versus ear), suggesting stable niche adaptation within a clonal lineage. This has provided an opportunity to identify determinants of disease tropism. Genomic analysis identified 17 and 27 single nucleotide polymorphisms (SNPs) or insertions/deletions in protein coding sequences between blood and ear isolates of serotype 14 ST15 and serotype 3 ST180, respectively. SNPs in raffinose uptake and utilization genes (rafR or rafK) were detected in both serotypes/lineages. Ear isolates were consistently defective in growth in media containing raffinose as the sole carbon source, as well as in expression of raffinose pathway genes aga, rafG, and rafK, relative to their serotype/ST-matched blood isolates. Similar differences were also seen between serotype 23F ST81 blood and ear isolates. Analysis of rafR allelic exchange mutants of the serotype 14 ST15 blood and ear isolates demonstrated that the SNP in rafR was entirely responsible for their distinct in vitro phenotypes and was also the determinant of differential tropism for the lungs versus ear and brain in a mouse intranasal challenge model. These data suggest that the ability of pneumococci to utilize raffinose determines the nature of disease.IMPORTANCES. pneumoniae is a component of the commensal nasopharyngeal microflora of humans, but from this reservoir, it can progress to localized or invasive disease with a frequency that translates into massive global morbidity and mortality. However, the factors that govern the switch from commensal to pathogen, as well as those that determine disease tropism, are poorly understood. Here we show that capacity to utilize raffinose can determine the nature of the disease caused by a given pneumococcal strain. Moreover, our findings provide an interesting example of convergent evolution, whereby pneumococci belonging to two unrelated serotypes/lineages exhibit SNPs in separate genes affecting raffinose uptake and utilization that correlate with distinct pathogenic profiles in vivo This further underscores the critical role of differential carbohydrate metabolism in the pathogenesis of localized versus invasive pneumococcal disease.

Effects of azelastine nasal spray on nasal and nasopharyngeal microflora.

Azelastine nasal spray is a topical antihistaminic drug for the symptomatic treatment of allergic rhinitis. This study aimed to investigate the effects of azelastine on nasal and nasopharyngeal microflora. Swab samples from 25 patients prescribed azelastine nasal spray monotherapy were collected just before treatment and after 1 month of treatment. After incubation of inoculates, the number of bacteria present in cultures was measured (in colony-forming units per millilitre). Evaluation of the number of microflora revealed increased bacterial reproduction after treatment, but this difference was not statistically significant. The use of azelastine nasal spray decreased the reproduction of three potentially pathogenic bacteria; however, it did not affect the reproduction of other potentially pathogenic bacteria. The use of azelastine nasal spray for one month did not have a statistically significant effect on the numbers of nasal and nasopharyngeal microflora; it is therefore safe from a microbiological viewpoint.

Representation of Streptococcus Pneumoniae in Outpatient Population of Sarajevo Canton.

Introduction:Streptococcus pneumoniae in asymptomatic manner colonize the mucous membranes of the nasopharynx of children and adults, but can cause serious illness in the media which are naturally sterile. In 5-40% of healthy population this bacteria colonize the nasopharyngeal mucosa thanks to the surface adhesin protein, which allow the bacteria to attach to the epithelial cells. The normal nasopharyngeal microflora retains pneumococcus in a small number and does not allow it to express its pathogenic potential and cause disease. If this dominance of the normal microflora is violated, after adherence and local duplication, pneumococcus can spread to the middle ear, sinuses or lungs. Colonization is more common in children than in adults.Goal:The goal of this study was to determine the prevalence of the carrier state and susceptibility of pneumococcal strains that circulate in the outpatient population of Sarajevo Canton as a potential source of infection.Material and methods:In the microbiological laboratory of the Institute of Public Health of Canton Sarajevo in the period from July 1, 2013 until April 15, 2014 were analyzed swabs of the nose and nasopharynx, eye and ear from a total of 4109 outpatients. Swabs were inoculated on blood agar nutrient medium. Then was performed catalase test, preparation by Gram and susceptibility test on Optochin. Isolates positive for S. pneumoniae were subjected to in vitro assays to investigate the antimicrobial susceptibility/resistance.Results:Out of 4109 analyzed swabs the pneumococcus positive was 180 (4.38%). Of these, 137 (76.11%) nasal and nasopharyngeal swabs, 33 (18.33%) of the eyes and 10 (5.56%) ear. The highest number of positive swabs were isolated in children aged 6 years and less, a total of 168 (93.33%), in children aged 7-13 years were positive 7 (3.89%), while among respondents aged 14-20 years only 5 (2.78%).Conclusions:The most common site for isolation of pneumococci is the nose and throat, and the most common carriers of these bacteria are children under 6 years of age. Determining the prevalence of the carrier state contributes to the improvement of preventive measures to reduce the risk of infection and possible sequels.

Dynamic changes in the Streptococcus pneumoniae transcriptome during transition from biofilm formation to invasive disease upon influenza A virus infection.

Streptococcus pneumoniae is a leading cause of infectious disease globally. Nasopharyngeal colonization occurs in biofilms and precedes infection. Prior studies have indicated that biofilm-derived pneumococci are avirulent. However, influenza A virus (IAV) infection releases virulent pneumococci from biofilms in vitro and in vivo. Triggers of dispersal include IAV-induced changes in the nasopharynx, such as increased temperature (fever) and extracellular ATP (tissue damage). We used whole-transcriptome shotgun sequencing (RNA-seq) to compare the S. pneumoniae transcriptome in biofilms, bacteria dispersed from biofilms after exposure to IAV, febrile-range temperature, or ATP, and planktonic cells grown at 37°C. Compared with biofilm bacteria, actively dispersed S. pneumoniae, which were more virulent in invasive disease, upregulated genes involved in carbohydrate metabolism. Enzymatic assays for ATP and lactate production confirmed that dispersed pneumococci exhibited increased metabolism compared to those in biofilms. Dispersed pneumococci also upregulated genes associated with production of bacteriocins and downregulated colonization-associated genes related to competence, fratricide, and the transparent colony phenotype. IAV had the largest impact on the pneumococcal transcriptome. Similar transcriptional differences were also observed when actively dispersed bacteria were compared with avirulent planktonic bacteria. Our data demonstrate complex changes in the pneumococcal transcriptome in response to IAV-induced changes in the environment. Our data suggest that disease is caused by pneumococci that are primed to move to tissue sites with altered nutrient availability and to protect themselves from the nasopharyngeal microflora and host immune response. These data help explain pneumococcal virulence after IAV infection and have important implications for studies of S. pneumoniae pathogenesis.

Streptococcus pneumoniae evades human dendritic cell surveillance by pneumolysin expression

Dendritic cells (DCs) protect the respiratory epithelium via induction of innate immune responses and priming of naïve T cells during the initiation of adaptive immunity. Streptococcus pneumoniae, a commonly carried asymptomatic member of the human nasopharyngeal microflora, can cause invasive and inflammatory diseases and the cholesterol-dependent cytotoxin pneumolysin is a major pneumococcal virulence factor implicated in compounding tissue damage and mediating inflammatory responses. While most studies examining the impact of pneumolysin have been based on murine models, we have focused this study on human DC responses. We show that expression of haemolytic pneumolysin inhibits human DC maturation, induction of proinflammatory cytokines and activation of the inflammasome. Furthermore, intracellular production of pneumolysin induces caspase-dependent apoptosis in infected DCs. Similarly, clinical isolates with non-haemolytic pneumolysin were more proinflammatory and caused less apoptosis compared to clonally related strains with active pneumolysin. This study describes a novel role of pneumolysin in the evasion of human DC surveillance that could have a profound clinical impact upon inflammatory disease progression and highlights the need to study human responses to human-specific pathogens.

Supplementation with the probiotic Lactobacillus casei Shirota has no immunomodulatory effect in healthy men with a low baseline NK cell activity

Probiotics have been shown to modulate the immune system. In healthy humans, however, contradictory results were observed. Thus, in a randomized double‐blind, placebo‐controlled trial the effect of the probiotic Lactobacillus casei Shirota (LcS; 1,95x1010 cfu/day) on immune functions of healthy men with low baseline NK cell activity (n=34/group) was investigated (study period 8 wk, i.e. a 2‐wk run‐in period, 4 wk supplementation and a‐2 wk follow‐up). Individuals of the control group (n=34) consumed the same drink manufactured without LcS. LcS had no effect on NK cell activity and immune cell phenotypes in peripheral blood mononuclear cells. Further, phagocytic activity and intensity of monocytes and neutrophils, oxidative burst, cytokine secretion pattern and saliva SIgA concentrations did not differ between groups. The incidence of potentially pathogenic microorganisms including S. aureus and hemolytic streptococci in the nasopharyngeal microflora was also not changed. In summary, under the present conditions supplementation with LcS caused no immunomodulation. However, other dosages of LcS might be effective as indicated by trials using higher LcS dosages. Dietary habits of study subjects may also be relevant for the outcome of the trial and may impede a demonstration of immunomodulatory activities of probiotics in open studies.The study was supported in part by a grant from Yakult Honsha CO., Ltd.

Methodology and Transport Medium for Collection of Helicobacter pylori on a String Test in Remote Locations

Helicobacter pylori can be isolated from patients using the string test but contaminating oral and nasopharyngeal microflora need to be suppressed by rapid plating out onto selective culture media. Recently, use of this diagnostic method was enhanced by using a novel transport medium to collect specimens from subjects in a remote Australian clinic over 1300 km from the laboratory. Retrieved string tests were transported to the laboratory in chilled polystyrene boxes in 5 ml screw-cap bottles with 3 ml of a brain heart infusion broth plus antibiotics. These were 20 g/ml vancomycin, 10 g/ml trimethoprim, 10 g/ml cefsulodin, and 10 g/ml amphotericin B. A comparison was made between subjects who gargled with a chlorhexidine mouthwash before swallowing the string test and those who did not. Forty-five urea breath test-positive subjects were tested and H. pylori was isolated from 34 of them. Successful culture was achieved from string tests that were in transit for up to 29 hours and where the maximum temperature in the transport box was 14 degrees C. The additional use of a mouthwash had a marked effect on the isolation rate. H. pylori was cultured from 75% of subjects who gargled but only from 39% who did not. This methodology and transport medium can broaden the use of the string test to more remote geographic areas where endoscopy is not feasible so that H. pylori isolates may still be obtained for diagnostic and epidemiologic studies. The value of this promising methodology of collection and transport should be assessed in a controlled study.

Investigation about the homogeneity of nasopharyngeal microflora at the different location of nasopharynx of children with acute otitis media

Nasopharynx is thought to be a very important site as becterial reservoir for acute otitis media (AOM). In this study, we investigated on the homogeneity of nasopharyngeal microflora at the different location of nasopharynx of children with AOM. Thirty nasopharyngeal samples of 15 children with AOM, two samples harvested from both nostrils of each child, were cultured and analyzed by patterns of antibiotic susceptibility and pulsed-field gel electrophoresis analysis (PFGE). A total of 30 nasopharyngeal samples were cultured and 19 isolates of Streptococcus pneumoniae from 10 children (66.7%), 8 isolates of Haemophilus influenzae from 4 children (26.7%), and 12 isolates of Moraxella catarrhalis from 7 children (46.7%) were obtained. In all children except three, the nasopharyngeal microflora at right and left orifice of the eustachian tubes showed no obvious differences in the bacterial species and quantities. Furthermore, in children with the same species of were cultured from right and left orifice of the eustachian tubes at the same time, all nine couples of S. pneumoniae isolates, four couples of H. influenzae isolates, and five couples of M. catarrhalis isolates showed about the same susceptibility and PFGE patterns. These results suggest that the microflora at the different location of nasopharynx of children with AOM is almost homogeneous, irrespective of the clinical signs.

In vitro investigation of the indirect pathogenicity of beta-lactamase-producing microorganisms in the nasopharyngeal microflora

Nasopharyngeal microflora contains some beta-lactamase-producing microorganisms. In this study, we investigated in vitro on the indirect pathogenicities of Haemophilus parainfluenzae (H. parainfluenzae) and Moraxella catarrhalis (M. catarrhalis) against the antipneumococcul activities of some beta-lactams. We compared the antimicrobial and bactericidal activities of beta-lactams against penicillin-susceptible Streptococcus pneumoniae (PSSP) with or without presence of the enzymes of two species of beta-lactamase-producing microorganisms, H. parainfluenzae and M. catarrhalis. When adding the enzymes extracted from these two beta-lactamase-producing microorganisms in equivalent amounts of 10(7) CFU/spot, the minimum inhibitory concentrations of amoxicillin (AMPC) and cefaclor (CCL) increased to >64 microg/mL. Even third-generation cephalosporins, such as cefditren (CDTR) and ceftriaxone (CTRX) showed marked increases with the enzyme of M. catarrhalis. In time-kill kinetics, same phenomenon was observed in mixed culture indicating the indirect pathogenicities of distinct bacteria, not extracted enzymes, on the cidal activities of beta-lactams against PSSP. Clavulanic acid (CVA)/AMPC, faropenem (FRPM), and imipenem (IPM) were not affected by these beta-lactamase-producing strains with respect to their activities against PSSP. However, these two beta-lactamase-producing strains and their enzymes did not show any significant influence on the antipneumococcul activities of beta-lactams, until the number of bacterial cells reached >10(8) CFU/mL. Our results suggest that these two species of beta-lactamase-producing microorganisms in the nasopharyngeal microflora may act as indirect pathogens on the antipneumococcul activities of beta-lactams with reflecting their substrate profiles, but this is dependent on sufficient amounts of enzyme for their influence as indirect pathogens.

Nasopharyngeal carriage of anaerobes during health and acute otitis media by two years of age

The nasopharyngeal acquisition of anaerobic bacteria was longitudinally examined among a homogeneous group of Caucasian infants by 2 years of age. Nasopharyngeal swab (NP) samples were collected at scheduled healthy visits at 2, 6, 12, 18, and 24 months of age, and nasopharyngeal aspirates (NPA) during every visit for acute otitis media (AOM). The infants were divided into 4 groups according to the number (0, 1, 2-3, and > 3) of AOM episodes experienced by 2 years of age. At 2 years, the cumulative carriage rates of anaerobic species in these infant groups were 29%, 62%, 89%, and 89%, respectively. Anaerobic species were found in 15/220 (6.8%) of the NP samples and in 34/71 (47.9%) of the NPA samples. Our present results indicate that anaerobic species do not belong to the indigenous nasopharyngeal microflora but only transiently colonize the nasopharynx during AOM.

Ecological effects on the oro- and nasopharyngeal microflora in children after treatment of acute otitis media with cefuroxime axetil or amoxycillin-clavulanate as suspensions

To evaluate if the extent of normal microflora disturbances differed between treatment with amoxycillin-clavulanate administered in an active form and cefuroxime axetil administered as an inactive prodrug. Twenty-eight children, 0.5-5 years old, diagnosed with acute otitis media (AOM), were treated with either amoxycillin-clavulanate (13.3 mg/kg 3 times daily) or cefuroxime axetil (15 mg/kg twice daily) for 7 days. Saliva samples and nasopharyngeal swabs were collected before, directly after and 2 weeks after treatment. The saliva samples were quantitatively and qualitatively analyzed and the nasopharyngeal swabs were qualitatively analyzed. All isolated strains were tested for beta-lactamase production. Both treatment regimens gave rise to similar alterations of the normal oropharyngeal microflora. In both groups, the amount of Streptococcus salivarius was significantly reduced (P < 0.05). The most common causative pathogens of acute otitis were S. pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. On the day of enrollment, approximately half of the patients, in both groups, were infected with more than one pathogen. The rate of infection or colonization with more than one potential pathogen was low on day 7 but recurred 2 weeks after treatment to similar levels as on day 0. The total number of patients with reinfection, recolonization or recurrence of pathogens on day 21 was 11/12 in the amoxycillin-clavulanate group and 4/7 in the cefuroxime axetil group. The most common beta-lactamase producer was M. catarrhalis. The local high concentration of antibiotics in the oropharynx immediately after intake of antibiotic suspensions seem to have little or no impact on the extent of disturbance of the microflora in this region. Children of this age group seem prone to either reinfection, recolonization or persistence of pathogens within 2 weeks after treatment. Furthermore, co-infection with more than one pathogen seems common in children with AOM and infection with beta-lactamase producing microorganisms occurs frequently.

Ribotyping of Strains of Moraxella (Branhamella) Catarrhalis Cultured from the Nasopharynx and Middle Ear of Children with Otitis Media

Moraxella (Branhaomella) catarrhalis is frequently present in the nasopharyngeal microflora of small children, especially during episodes of acute otitis media . By means of ribotyping (restriction endonuclease analysis of chromosomal DNA combined with rRNA probing), we studied the genetic heterogeneity of 78 cultures of M. catarrhalis obtained from different localities in the nasopharynx of nine young children with secretory otitis media. Using HindIII and PstI as endonucleases, five different ribotypes were recognized, representing at least five different genotypes of M. catarrhalis. The distribution of these types was found to be almost identical to the distribution among 16 M. catarrhalis strains cultured from middle ear exudates of 16 children with acute otitis media. Ribotype HAPA was found in two-thirds of all the cultures investigated, and 44% of the children harboured more than one ribotype in the nasopharynx at the same time. The vast majority of the nasopharyngeal M. catarrhalis cultures were beta-lactamase positive. One child had both a HAPA ribotype, beta-lactamase-negative strain in the nasopharyngeal secretions, and HAPA ribotype, beta-lactamase-positive strains at the entrance of the eustachian tube, the nasopharyngeal tonsils, the folds of the nasopharyngeal tonsils and the oropharynx. All except one of the M. catarrhalis strains cultured from middle ear exudates were beta-lactamase positive.