Inflammatory responses and barrier disruption in the trachea of chicks following Mycoplasma gallisepticum infection: a focus on the TNF-α-NF-κB/MLCK pathway.

Mycoplasma gallisepticum (MG) can induce persistent inflammatory damage to the tracheal mucosa of poultry and cause chronic respiratory diseases in chickens. To further investigate the mechanism of MG-induced injury to the tracheal mucosa, we used chick embryo tracheal organ culture (TOC) as a model to study the invasion and reproduction of MG, the effect of MG on tracheal morphology, and the potential factors that promote MG tissue invasion. The results showed that MG infection significantly damaged the tracheal epithelial structure and weakened tracheal epithelial barrier function; MG also increased the occurrence of bacterial displacement, with a significant (p < 0.05) increase in the bacterial load of the infected TOCs at 5 and 7 days post-infection. In addition, MG significantly (p < 0.05) increased the expression levels of inflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß), and IL-6, and activated the NF-κB signalling pathway, leading to increased nuclear translocation of NF-κB p65. Simultaneously, the map kinase pathway (MAPK) was activated. This activation might be associated with increased myosin light chain (MLC) phosphorylation, which could lead to actin-myosin contraction and disruption of tight junction (TJ) protein function, potentially compromising epithelial barrier integrity and further catalysing MG migration into tissues. Overall, our results contribute to a better understanding of the interaction between MG and the host, provide insight into the mechanisms of damage to the tracheal mucosa induced by MG infection, and provide new insights into the possible pathways involved in Mycoplasma gallisepticum infection in vivo.

Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa.

BACKGROUND: The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. METHODS: Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. RESULTS: The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95-100% similarity to the isolates from the gene bank. CONCLUSION: The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

COMPARISON OF TRACHEAL ASPIRATES IN THE PERIOD BEFORE AND AFTER THE START OF THE COVID-19 PANDEMIC IN THE INTENSIVE CARE UNIT IN A TERTIARY HOSPITAL.

Changes in working methods and diagnostics using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) diagnostics that occurred after the start of the COVID-19 pandemic could show differences in the prevalence of positive microbiological samples. In a retrospective study, a total of 442 tracheal aspirates in the pre-pandemic period (Period A, 2018, 198 patients, age median 69 (57-78)) and 277 samples in the pandemic period (Period B, 2021, 147 patients, age 68 (56-77) (p=0.585) obtained after the start of the pandemic were analyzed. A total of 176 patients had at least 1 positive result. In Period A, there were 245 (55%) and in Period B 186 (68%) sterile samples (p=0.001). The most frequently isolated pathogens were Acinetobacter baumannii in 86 patients from Period A and 32 patients from Period B, i.e., 43% vs. 21.7% of all positive isolates (p=0.247), followed by Pseudomonas aeruginosa in 29 patients in Period A (14.6%) vs. 7 (3%) (p=0.112) in Period B. A statistically significant increase was observed in the incidence of Enterobacterales (16.6% vs. 32.6%, p=0.002), especially Klebsiellae spp. Although overall mortality decreased in Period B, changes in the working methods and diagnostics did not result in changes in the mortality of patients whose tracheal aspirates were sampled.

Corynebacterium zhongnanshanii sp. nov. isolated from trachea of Marmota himalayana, Corynebacterium lujinxingii sp. nov. and Corynebacterium wankanglinii sp. nov. from human faeces.

Six novel facultatively anaerobic, Gram-stain-positive, rod-shaped, non-haemolytic bacteria (zg-320T/zg-336, zg-917T/zg-910 and zg-913T/zg-915) isolated from animal tissues and human faeces were found to belong to the genus Corynebacterium based on the phylogenetic analyses of 16S rRNA gene and 262 core genes set. Based on the greatest degree of 16S rRNA similarity, zg-320T/zg-336 had the highest 16S rRNA gene similarity to Corynebacterium falsenii DSM 44353T (97.51 %), zg-917T/zg-910 to Corynebacterium coyleae DSM 44184T (98.68 %), and zg-913T/zg-915 to Corynebacterium afermentans subsp. lipophilum CIP 103500T (98.79 %). The three novel type strains had a relatively high DNA G+C content (61.2-64.4 mol%), low DNA relatedness and ANI values with their respective neighbours: 23.5/72.7 %, 25.0/72.3%and 22.6/73.1 % (zg-320T vs. Corynebacterium auriscanis CIP 106629T, Corynebacterium resistens DSM 45100T and Corynebacterium suicordis DSM 45110T); 24.4/82.3% and 23.7/81.3 % (zg-917T vs. C. coyleae DSM 44184T and Corynebacterium jeddahense JCBT); 26.8/83.7% and 27.7/84.4 % (zg-913T vs. Corynebacterium mucifaciens ATCC 700355T and C. afermentans subsp. lipophilum CCUG 32105T). The three novel species had C16 : 0, C18 : 0, C18 : 1 ω9c and C18 : 0 ante/C18 : 2 ω6,9c as the major cellular fatty acids; MK-8(H2) in strain zg-917T and MK-9(H2) in strains zg-320T and zg-913T were found to be the major respiratory quinones. For the three novel species, the detected major polar lipids included diphosphatidylglycerol, phosphatidyl inositol mannoside, phosphatidylglycerol and phosphatidylinositol, the cell-wall peptidoglycan was based on meso-DAP, and the whole-cell sugars mainly included ribose, arabinose and galactose. The three novel species grew optimally at 35-37 °C, 0.5 % (w/v) NaCl and pH 7.0-8.0; notably, they were tolerant of 10.5 % (w/v) NaCl. Based on the results of these comprehensive analyses, three novel species in the genus Corynebacterium are proposed, aptly named Corynebacterium zhongnanshanii sp. nov. (zg-320T = GDMCC 1.1719T = JCM 34106T), Corynebacterium lujinxingii sp. nov. (zg-917T = GDMCC 1.1707T = JCM 34094T) and Corynebacterium wankanglinii sp. nov. (zg-913T = GDMCC 1.1706T = JCM 34398T).

Accuracy of galactomannan testing on tracheal aspirates in COVID-19-associated pulmonary aspergillosis.

OBJECTIVE: Our aim was to evaluate the performance of two galactomannan (GM) assays (Platelia Aspergillus EIA, Bio-Rad® , and Aspergillus GM LFA, IMMY® ) in tracheal aspirate (TA) samples of consecutive critically ill patients with COVID-19. METHODS: We included critically ill patients, performed GM-EIA and GM-Lateral Flow Assay (GM-LFA) in TA and followed them until development of COVID-19-associated pulmonary aspergillosis (CAPA) or alternate diagnosis. CAPA was defined according to the modified AspICU criteria in patients with SARS-CoV-2 infection. We estimated sensitivity, specificity, positive and negative predictive values for GM-EIA, GM-LFA, the combination of both or either positive results for GM-EIA and GM-LFA. We explored accuracy using different breakpoints, through ROC analysis and Youden index to identify the optimal cut-offs. We described antifungal treatment and 30-day mortality. RESULTS: We identified 14/144 (9.7%) patients with CAPA, mean age was 50.35 (SD 11.9), the median time from admission to CAPA was 8 days; 28.5% received tocilizumab and 30-day mortality was 57%. ROC analysis and Youden index identified 2.0 OD as the best cut-off, resulting in sensitivity and specificity of 57.1% and 81.5% for GM-EIA and 60% and 72.6% for GM-LFA, respectively. CONCLUSIONS: The diagnostic performance of GM in tracheal aspirates improved after using a cut-off of 2 OD. Although bronchoalveolar lavage testing is the ideal test, centres with limited access to bronchoscopy may consider this approach to identify or rule out CAPA.

Differential Response of the Chicken Trachea to Chronic Infection with Virulent Mycoplasma gallisepticum Strain Ap3AS and Vaxsafe MG (Strain ts-304): a Transcriptional Profile.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Coinfection with Porcine Circovirus Type 2 (PCV2) and Streptococcus suis Serotype 2 (SS2) Enhances the Survival of SS2 in Swine Tracheal Epithelial Cells by Decreasing Reactive Oxygen Species Production.

Porcine circovirus type 2 (PCV2) and Streptococcus suis serotype 2 (SS2) clinical coinfection cases have been frequently detected. The respiratory epithelium plays a crucial role in host defense against a variety of inhaled pathogens. Reactive oxygen species (ROS) are involved in killing of bacteria and host immune response. The aim of this study is to assess whether PCV2 and SS2 coinfection in swine tracheal epithelial cells (STEC) affects ROS production and investigate the roles of ROS in bacterial survival and the inflammatory response. Compared to SS2 infection, PCV2/SS2 coinfection inhibited the activity of NADPH oxidase, resulting in lower ROS levels. Bacterial intracellular survival experiments showed that coinfection with PCV2 and SS2 enhanced SS2 survival in STEC. Pretreatment of STEC with N-acetylcysteine (NAC) also helps SS2 intracellular survival, indicating that PCV2/SS2 coinfection enhances the survival of SS2 in STEC through a decrease in ROS production. In addition, compared to SS2-infected STEC, PCV2/SS2 coinfection and pretreatment of STEC with NAC prior to SS2 infection both downregulated the expression of the inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1ß. Further research found that activation of p38/MAPK promoted the expression of inflammatory cytokines in SS2-infected STEC; however, PCV2/SS2 coinfection or NAC pretreatment of STEC inhibited p38 phosphorylation, suggesting that coinfection of STEC with PCV2 and SS2 weakens the inflammatory response to SS2 infection through reduced ROS production. Collectively, coinfection of STEC with PCV2 and SS2 enhances the intracellular survival of SS2 and weakens the inflammatory response through decreased ROS production, which might exacerbate SS2 infection in the host.

Presence of Gastric Pepsinogen in the Trachea Is Associated with Altered Inflammation and Microbial Composition.

Gastroesophageal reflux is a common gastrointestinal issue that can lead to aspiration and contribute to respiratory problems. Little is known about how reflux can alter the respiratory microenvironment. We aimed to determine if the presence of gastric pepsinogen in the trachea was associated with changes in the microbial and inflammatory microenvironment. A pediatric cohort at high risk of reflux aspiration was prospectively recruited, and the tracheal microenvironment was examined. Pepsinogen A3 (PGA3) and cytokines were measured. The microbiome (bacterial and fungal) was profiled using 16S rRNA and internal transcribed spacer 2 (ITS2) amplicon sequencing. Increased bacterial richness and an altered composition driven by an enrichment of Prevotella correlated with high PGA3 levels. Fungal richness increased with PGA3, with higher Candida relative abundances observed in a subset of samples with high PGA3 levels. Source tracking of tracheal microbial taxa against taxa from matched oral and gastric samples revealed a significantly greater contribution of oral than of gastric taxa with higher PGA3 levels. Tracheal cytokines were differentially produced when stratified according to PGA3, with higher levels of interleukin-1 (IL-1)-related cytokines and IL-8 being associated with high PGA3 levels. Network analysis across cytokine and microbiome measures identified relationships between IL-1-related proteins and microbial taxa, with the presence of respiratory issues associated with higher levels of IL-1ß, IP-10, and Prevotella In conclusion, PGA3 levels in the trachea are correlated with increases in specific microbial taxa and inflammatory molecules, with an increase in oral microbes with increasing PGA3.

Therapeutic Inhaled Sphingosine for Treating Lung Infection in a Mouse Model of Critical Illness.

BACKGROUND/AIMS: Sphingosine, a sphingoid long chain base, is a natural lipid with antimicrobial properties. Recent animal studies have shown that preventive sphingosine inhalation can rescue susceptible mice, such as cystic fibrosis-, burn injured- or aged mice from bacterial pulmonary infection. While preventing lung infections in susceptible patients has obvious clinical merit, treatment strategies for an established infection are also direly needed, particularly in the times of rising antibiotic resistance. Here, we tested the potential of sphingosine in treating an established pulmonary infection. METHODS: We used a cecal ligation and puncture (CLP) model in male CF-1 mice and a Pseudomonas aeruginosa strain that was isolated from a septic patient (P. aeruginosa 762). We determined susceptibility to intranasal infection and ascertained when the pulmonary infection was established by continuous core body temperature monitoring. We quantified sphingosine levels in the tracheal epithelium by immunohistochemistry and studied the effects on sphingosine on bacterial membrane permeabilization and intracellular acidification using fluorescent probes. RESULTS: We firstdetermined that septic mice are highly susceptible to P. aeruginosa infection 2 days after indu-cing sepsis. Additionally, at this time, sphingosine levels in the tracheal epithelium are significantly reduced as compared to levels in healthy mice. Secondly, upon intranasal Pseudomonasinoculation, we ascertained that pulmonary infection was established as early as 2.5 h after inoculation as evidenced by a significant drop in core body temperature. Using these times of infection susceptibility and detection (2 days post CLP, 2.5h after inoculation) we treated with inhaled sphingosine and observed pulmonary bacterial loads reduced to levels found in infected healthy mice after inoculation and decreased infection-associated mortality. Further, our data demonstrate that sphingosine induces outer membrane permeabilization, disrupting the membrane potential and leading to intracellular acidification of the bacteria. CONCLUSION: Sphingosine shows efficacy in treating P. aeruginosa lung infections not only prophylactically, but also therapeutically.

Dissimilarity of the gut-lung axis and dysbiosis of the lower airways in ventilated preterm infants.

BACKGROUND: Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth, but the role of the microbiome in its development remains unclear. Therefore, we assessed the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways, and assessed the gut-lung axis by comparing bacterial communities in the upper and lower airways with stool findings. Finally, we assessed whether the bacterial communities were associated with lung inflammation to suggest dysbiosis. METHODS: We serially sampled multiple anatomical sites including the upper airway (nasopharyngeal aspirates), lower airways (tracheal aspirate fluid and bronchoalveolar lavage fluid) and the gut (stool) of ventilated preterm-born infants. Bacterial DNA load was measured in all samples and sequenced using the V3-V4 region of the 16S rRNA gene. RESULTS: From 1102 (539 nasopharyngeal aspirates, 276 tracheal aspirate fluid, 89 bronchoalveolar lavage, 198 stool) samples from 55 preterm infants, 352 (32%) amplified suitably for 16S RNA gene sequencing. Bacterial load was low at birth and quickly increased with time, but was associated with predominant operational taxonomic units (OTUs) in all sample types. There was dissimilarity in bacterial communities between the upper and lower airways and the gut, with a separate dysbiotic inflammatory process occurring in the lower airways of infants. Individual OTUs were associated with increased inflammatory markers. CONCLUSIONS: Taken together, these findings suggest that targeted treatment of the predominant organisms, including those not routinely treated, such as Ureaplasma spp., may decrease the development of CLD in preterm-born infants.

Respiratory and Gut Microbiota in Commercial Turkey Flocks with Disparate Weight Gain Trajectories Display Differential Compositional Dynamics.

Communities of gut bacteria (microbiota) are known to play roles in resistance to pathogen infection and optimal weight gain in turkey flocks. However, knowledge of turkey respiratory microbiota and its link to gut microbiota is lacking. This study presents a 16S rRNA gene-based census of the turkey respiratory microbiota (nasal cavity and trachea) alongside gut microbiota (cecum and ileum) in two identical commercial Hybrid Converter turkey flocks raised in parallel under typical field commercial conditions. The flocks were housed in adjacent barns during the brood stage and in geographically separated farms during the grow-out stage. Several bacterial taxa, primarily Staphylococcus, that were acquired in the respiratory tract at the beginning of the brood stage persisted throughout the flock cycle. Late-emerging predominant taxa in the respiratory tract included Deinococcus and Corynebacterium Tracheal and nasal microbiota of turkeys were identifiably distinct from one another and from gut microbiota. Nevertheless, gut and respiratory microbiota changed in parallel over time and appeared to share many taxa. During the brood stage, the two flocks generally acquired similar gut and respiratory microbiota, and their average body weights were comparable. However, there were qualitative and quantitative differences in microbial profiles and body weight gain trajectories after the flocks were transferred to geographically separated grow-out farms. Lower weight gain corresponded to the emergence of Deinococcus and Ornithobacterium in the respiratory tract and Fusobacterium and Parasutterella in gut. This study provides an overview of turkey microbiota under field conditions and suggests several hypotheses concerning the respiratory microbiome.IMPORTANCE Turkey meat is an important source of animal protein, and the industry around its production contributes significantly to the agricultural economy. The microorganisms present in the gut of turkeys are known to impact bird health and flock performance. However, the respiratory microbiota in turkeys is entirely unexplored. This study has elucidated the microbiota of respiratory tracts of turkeys from two commercial flocks raised in parallel throughout a normal flock cycle. Further, the study suggests that bacteria originating in the gut or in poultry house environments influence respiratory communities; consequently, they induce poor performance, either directly or indirectly. Future attempts to develop microbiome-based interventions for turkey health should delimit the contributions of respiratory microbiota and aim to limit disturbances to those communities.

Streptococcus suis suilysin compromises the function of a porcine tracheal epithelial barrier model.

Streptococcus suis is a bacterial pathogen that mainly colonizes the upper respiratory tract of pigs. It is known to cause severe infections such as septicemia, meningitis, arthritis, and endocarditis in pigs and to be responsible for major economic losses in the swine industry worldwide. To better understand the interactions between S. suis and the porcine respiratory epithelium, we investigated the ability of this pathogen to cause damage to the tracheal epithelial barrier. We showed that S. suis compromises the integrity of a tracheal epithelial barrier model as determined by measuring transepithelial electrical resistance and paracellular flux of FITC-dextran. As a consequence of this breakdown, S. suis translocates across the epithelial cell monolayer. On the other hand, a S. suis mutant deficient in the production of suilysin, a cholesterol-dependent cytolysin, was significantly impaired in its ability to cause damage to the epithelial barrier. In addition, a recombinant suilysin disrupted the integrity of the tracheal epithelial barrier. Immunofluorescence staining suggested that suilysin affects two major tight junction proteins (occludin and zonula occludens-1). In summary, S. suis is able to compromise the function of the porcine respiratory epithelial barrier through the action of suilysin. This better knowledge of the interactions between S. suis and tracheal epithelial cells may help in the development of novel strategies to prevent the invasion of the epithelium by this and other swine respiratory pathogens.

Psychrobacter pygoscelis sp. nov. isolated from the penguin Pygoscelis papua.

One slightly beige-white pigmented, Gram-stain-negative, rod-shaped bacterium, strain I-STPP5bT, was isolated from the trachea of a Gentoo penguin chick individual (Pygoscelin papua) investigated in Fildes Bay, Chilean Antarctic (62° 12' S, 58° 57' W). I-STPP5bT consists of a 3.4 Mb chromosome with a DNA G+C content of 44.4 mol%. Of the 3056 predicted genes, 1206 were annotated as hypothetical proteins and 51 were tRNAs. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the isolate shared a 16S rRNA gene sequence identity to the type strains of Psychrobacter phenylpyruvicus (98.8 %), Psychrobacter arenosus and Psychrobacter pasteurii (both 98.3 %), Psychrobacter piechaudii (98.2 %) and Psychrobacter sanguinis (98.1 %), but 16S rRNA gene sequence similarities to all other Psychrobacter species were ≤98.0 %. Partial gyrB nucleotide and amino acid sequence similarities among strain STPP5bT and the next related type strains were all below 81.8 and 92.9%, respectively. DNA-DNA hybridisation (DDH) with P. phenylpyruvicus LMG 5372T, P. arenosus DSM 15389T and P. sanguinis DSM 23635T also showed low values (all below 30 %). The main cellular fatty acids of the strain were C18 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c. Based on phylogenetic, chemotaxonomic, genomic and phenotypic analyses we propose a new species of the genus Psychrobacter, with the name Psychrobacter pygoscelis sp. nov. and strain I-STPP5bT (=CIP 111410T= CCM 8799T=LMG 30301T) as type strain.

Porcine circovirus type 2 exploits JNK-mediated disruption of tight junctions to facilitate Streptococcus suis translocation across the tracheal epithelium.

Porcine circovirus type 2 (PCV2) is considered as the primary pathogen of porcine circovirus-associated disease (PCVAD), which results in significant economic losses worldwide. Clinically, PCV2 often causes disease through coinfection with other bacterial pathogens, including Streptococcus suis (S. suis), and especially the highly prevalent S. suis serotype 2 (SS2). The present study determined that continuous PCV2 infection in piglets down-regulates tight junction proteins (TJ) ZO-1 and occludin in the lungs. Swine tracheal epithelial cells (STEC) were used to explore the mechanisms and consequences of disruption of TJ, and an in vitro tracheal epithelial barrier model was established. Our results show that PCV2 infection in STEC decreases the expression levels of ZO-1 and occludin and increases the permeability of the tracheal epithelial barrier, resulting in easier translocation of SS2. Moreover, Western blot analysis indicates that PCV2 infection activates the JNK/MAPK pathway. The disruption of TJ in SETC and increased permeability of the epithelial barrier induced by PCV2 could be alleviated by inhibition of JNK phosphorylation, which indicates that the JNK/MAPK pathway regulates the expression of ZO-1 and occludin during PCV2 infection. This study allows us to better understand the mechanisms of PCV2 coinfection with bacterial pathogens and provides new insight into controlling the occurrence of PCVAD.

Expression of small RNAs of Bordetella pertussis colonizing murine tracheas.

We performed RNA sequencing on Bordetella pertussis, the causative agent of whooping cough, and identified nine novel small RNAs (sRNAs) that were transcribed during the bacterial colonization of murine tracheas. Among them, four sRNAs were more strongly expressed in vivo than in vitro. Moreover, the expression of eight sRNAs was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. The present results suggest a BvgAS-independent gene regulatory system involving the sRNAs that is active during B. pertussis infection.

Invasive pseudomembranous upper airway and tracheal Aspergillosis refractory to systemic antifungal therapy and serial surgical debridement in an Immunocompetent patient.

BACKGROUND: The development of respiratory infections secondary to Aspergillus spp. spores found ubiquitously in the ambient environment is uncommon in immunocompetent patients. Previous reports of invasive upper airway aspergillosis in immunocompetent patients have generally demonstrated the efficacy of treatment regimens utilizing antifungal agents in combination with periodic endoscopic debridement, with symptoms typically resolving within months of initiating therapy. CASE PRESENTATION: A 43-year-old previously healthy female presented with worsening respiratory symptoms after failing to respond to long-term antibiotic treatment of bacterial sinusitis. Biopsy of her nasopharynx and trachea revealed extensive fungal infiltration and Aspergillus fumigatus was isolated on tissue culture. Several months of oral voriconazole monotherapy failed to resolve her symptoms and she underwent mechanical debridement for symptom control. Following transient improvement, her symptoms subsequently returned and failed to fully resolve in spite of increased voriconazole dosing and multiple additional tissue debridements over the course of many years. CONCLUSIONS: Invasive upper airway aspergillosis is exceedingly uncommon in immunocompetent patients. In the rare instances that such infections do occur, combinatorial voriconazole and endoscopic debridement is typically an efficacious treatment approach. However, some patients may continue to experience refractory symptoms. In such cases, continued aggressive treatment may potentially slow disease progression even if complete disease resolution cannot be achieved.

In vitro characterization and genetic diversity of Bordetella avium field strains.

Bordetella avium (BA) is a respiratory pathogen of particular importance for turkeys. Specific adherence and damage to the respiratory epithelia are crucial steps of the pathogenesis, but knowledge about the mechanisms and the variety of virulence in field strains is limited. We analysed 17 BA field strains regarding their in vitro virulence-associated properties in tracheal organ cultures (TOC) of turkey embryos, and their genetic diversity. The TOC adherence assay indicated that BA field strains differ considerably in their ability to adhere to the tracheal mucosa, while the TOC ciliostasis assay illustrated a high degree of diversity in ciliostatic effects. These two virulence-associated properties were associated with each other in the investigated strains. Three of the investigated strains displayed significantly (P > 0.05) lower in vitro virulence in comparison to other strains. Genetic diversity of BA strains was analysed by core genome multilocus sequence typing (cgMLST). We applied a cgMLST scheme comprising 2667 targets of the reference genome (77.3% of complete genome, BA strain 197N). The results showed a broad genetic diversity in BA field strains but did not demonstrate a correlation between sequence type and virulence-associated properties. The cgMLST analysis revealed that strains with less marked virulence-associated properties had a variety of mutations in the putative filamentous haemagglutinin gene. Likewise, amino acid sequence alignment indicated variations in the protein. The results from our study showed that both adherence and ciliostasis assay can be used for virulence characterization of BA. Variations in the filamentous haemagglutinin protein may be responsible for reduced virulence of BA field strains.

Acquisition and Development of the Extremely Preterm Infant Microbiota Across Multiple Anatomical Sites.

OBJECTIVES: Microbial communities influencing health and disease are being increasingly studied in preterm neonates. There exists little data, however, detailing longitudinal microbial acquisition, especially in the most extremely preterm (<26 weeks' gestation). This study aims to characterize the development of the microbiota in this previously under-represented cohort. METHODS: Seven extremely preterm infant-mother dyads (mean gestation 23.6 weeks) were recruited from a single neonatal intensive care unit. Oral and endotracheal secretions, stool, and breast milk (n = 157 total), were collected over the first 60 days of life. Targeted 16S rRNA gene sequencing identified bacterial communities present. RESULTS: Microbiota of all body sites were most similar immediately following birth and diverged longitudinally. Throughout the sampling period Escherichia, Enterococcus, Staphylococcus, and an Enterobacteriaceae were dominant and well dispersed across all sites. Temporal divergence of the stool from other microbiota was driven by decreasing diversity and significantly greater proportional abundance of Bifidobacteriaceae compared to other sites. CONCLUSIONS: Four taxa dominated all anatomical sampling sites. Rare taxa promoted dissimilarity. Cross-seeding between upstream communities and the stool was demonstrated, possibly relating to buccal colostrum/breast milk exposure and indwelling tubes. Given the importance of dysbiosis in health and disease of extremely preterm infants, better understanding of microbial acquisition within this context may be of clinical benefit.

Evaluation of semi-quantitative compared to quantitative cultures of tracheal aspirates for the yield of culturable respiratory pathogens - a cross-sectional study.

BACKGROUND: Diagnosis of lower respiratory tract infections (LRTI) depends on the presence of clinical, radiological and microbiological findings. Endotracheal suction aspirate (ETSA) is the commonest respiratory sample sent for culture from intubated patients. Very few studies have compared quantitative and semi-quantitative processing of ETSA cultures for LRTI diagnosis. We determined the diagnostic accuracy of quantitative and semi-quantitative ETSA culture for LRTI diagnosis, agreement between the quantitative and semi quantitative culture techniques and the yield of respiratory pathogens with both methods. METHODS: This was a cross-sectional study conducted at the Aga Khan University clinical laboratory, Karachi, Pakistan. One hundred and seventy-eight ETSA samples sent for routine bacteriological cultures were processed quantitatively as part of regular specimen processing method and semi-quantitatively. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy was calculated for both methods using clinical diagnosis of pneumonia as reference standard. Agreement between the quantitative and semi quantitative methods was assessed via the kappa statistic test. Pathogen yield between the two methods was compared using Pearson's chi-square test. RESULTS: The quantitative and semi-quantitative methods yielded pathogens in 81 (45.5%) and 85 (47.8%) cases respectively. There was complete concordance of both techniques in 155 (87.1%) ETSA samples. No growth was observed in 45 (25.3%) ETSA specimens with quantitative culture and 37 (20.8%) cases by semi-quantitative culture. The diagnostic accuracy of both techniques were comparable; 64.6% for quantitative and 64.0% for semi-quantitative culture. The kappa agreement was found to be 0.84 (95% CI, 0.77-0.91) representing almost perfect agreement between the two methods. Although semi-quantitative cultures yielded more pathogens (47.8%) as compared to quantitative ETSA cultures (45.5%), the difference was only 2.3%. However, this difference achieved statistical (chi-square p-value < 0.001) favoring semi-quantitative culture methods over quantitative culture techniques for processing ETSA. CONCLUSION: In conclusion, there is a strong agreement between the performances of both methods of processing ETSA cultures in terms of accuracy of LRTI diagnosis. Semi-quantitative cultures of ETSA yielded more pathogens as compared to quantitative cultures. Although both techniques were comparable, we recommend processing of ETSA using semi-quantitative technique due to its ease and reduced processing time.

The microbiome of the upper respiratory tract in health and disease.

The human upper respiratory tract (URT) offers a variety of niches for microbial colonization. Local microbial communities are shaped by the different characteristics of the specific location within the URT, but also by the interaction with both external and intrinsic factors, such as ageing, diseases, immune responses, olfactory function, and lifestyle habits such as smoking. We summarize here the current knowledge about the URT microbiome in health and disease, discuss methodological issues, and consider the potential of the nasal microbiome to be used for medical diagnostics and as a target for therapy.