Detection of bacterial DNA in synovial fluid in dogs with arthritis: a comparison between bacterial culture and 16S rRNA polymerase chain reaction.

BACKGROUND: Septic arthritis (SA) is a serious condition in dogs that requires a prompt diagnosis and treatment to minimize long-term joint pathology. Although bacterial detection in synovial fluid (SF) through culture or cytology is often performed to confirm diagnosis, the sensitivity of these tests is low. The need for a reliable diagnostic tool to confirm the presence of bacteria in SF in humans has led to the increased use of 16S rRNA (i.e., ribosomal RNA) gene sequencing by polymerase chain reaction (16S rRNA PCR). The aim of this prospective clinical study was to compare the sensitivity and specificity of 16S rRNA PCR with bacterial culture on blood agar plates after pre-incubation of SF in paediatric blood bacterial culture bottles to identify bacteria in dogs with clinical signs of SA and to investigate the usefulness of these methods as diagnostic tools. RESULTS: Ten dogs with clinical signs of SA, nine with osteoarthritis (OA, control group) and nine with clinical signs of immune-mediated polyarthritis (IMPA, second control group) were examined. Bacterial culture was positive in seven of 10 dogs with clinical SA, of which only two were positive by 16S rRNA PCR. The sensitivity of 16S rRNA PCR and bacterial culture analysis for dogs with clinical SA were 20% and 70%, respectively. All SF samples collected from control group (n = 9) and second control group (n = 14) animals were negative on culture, and 16S rRNA PCR rendered a specificity of 100%. CONCLUSIONS: Our study showed a lower sensitivity of 16S rRNA PCR than bacterial culture for dogs with clinical SA. Our findings suggest that there is currently no advantage in using 16S rRNA PCR as a diagnostic tool for dogs with clinical SA. Furthermore, our study indicates that pre-incubation in paediatric blood bacterial culture bottles before bacterial cultivation on blood agar plates might enhance bacterial culture sensitivity compared to other culture methods.

Lyme neuroborreliosis in Swedish children-PCR as a complementary diagnostic method for detection of Borrelia burgdorferi sensu lato in cerebrospinal fluid.

The aim of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for the detection of Borrelia burgdorferi s.l. in CSF of Swedish children with LNB. This study was performed retrospectively on CSF and serum samples collected from children evaluated for LNB (n = 233) and controls with other specific neurological disorders (n = 59) in a Swedish Lyme endemic area. For anti-Borrelia antibody index, the IDEIA Lyme Neuroborreliosis kit (Oxoid) was used. Two in-house real-time PCR assays targeting the 16S rRNA gene were evaluated (TaqMan® and LUX™). Among patients classified as LNB cases (n = 102), five children (5%) were Borrelia PCR-positive in CSF with the TaqMan® assay. In the Non-LNB group (n = 131), one patient was Borrelia PCR positive with the TaqMan® assay. Among controls (n = 59), all CSF samples were PCR negative. When amplifying and sequencing ospA, we found B. garinii (n = 2), B. afzelii (n = 2), B. bavariensis (n = 1), and one untypable (n = 1). With the LUX™ technology, all CSF samples were PCR negative. The TaqMan® assay could detect only few cases (n = 6) of B. burgdorferi s.l. in CSF among children with LNB and the sensitivity was very low (5%). However, using larger CSF volumes and centrifugation of samples, the PCR technique could still be useful as a complementary diagnostic method when evaluating LNB. Furthermore, detection of spirochete DNA in clinical matrices, including CSF, is the method of choice for studying epidemiological aspects of LNB, a tick-borne emerging disease.

Activity of airway antimicrobial peptides against cystic fibrosis pathogens.

Antimicrobial peptides are important players of the innate host defence against invading microorganisms. The aim of this study was to evaluate the activity of airway antimicrobial peptides against the common cystic fibrosis (CF) pathogen Pseudomonas aeruginosa, and to compare it to the emerging multi-drug resistant CF pathogens Achromobacter xylosoxidans and Stenotrophomonas maltophilia. Clinical bacterial isolates from CF patients were used, and the antimicrobial activity of human beta-defensin 2 and 3, LL37 and lysozyme was evaluated using radial diffusion assay and viable counts. The cell surface zeta potential was analysed to estimate the net charge at the bacterial surface. Of the bacterial species included in the study, A. xylosoxidans was the most resistant to antimicrobial peptides, whereas P. aeruginosa was the most susceptible. The net charge of the bacterial surface was significantly more negative for P. aeruginosa compared to A. xylosoxidans, which may in part explain the differences in susceptibility.

Morbidity and mortality in critically ill patients with invasive group A streptococcus infection: an observational study.

BACKGROUND: Group A streptococci (GAS) are known to cause serious invasive infections, but little is known about outcomes when patients with these infections are admitted to intensive care. We wanted to describe critically ill patients with severe sepsis or septic shock due to invasive GAS (iGAS) and compare them with other patients with severe sepsis or septic shock. METHODS: Adult patients admitted to a general intensive care unit (ICU) in Sweden (2007-2019) were screened for severe sepsis or septic shock according to Sepsis 2 definition. Individuals with iGAS infection were identified. The outcome variables were mortality, days alive and free of vasopressors and invasive mechanical ventilation, maximum acute kidney injury score for creatinine, use of continuous renal replacement therapy and maximum Sequential Organ Failure Assessment score during the ICU stay. Age, Simplified Acute Physiology Score (SAPS 3) and iGAS were used as independent, explanatory variables in regression analysis. Cox regression was used for survival analyses. RESULTS: iGAS was identified in 53 of 1021 (5.2%) patients. Patients with iGAS presented a lower median SAPS 3 score (62 [56-72]) vs 71 [61-81]), p <  0.001), had a higher frequency of cardiovascular cause of admission to the ICU (38 [72%] vs 145 [15%], p <  0.001) and had a higher median creatinine score (173 [100-311] vs 133 [86-208] µmol/L, p <  0.019). Of the GAS isolates, 50% were serotyped emm1/T1 and this group showed signs of more pronounced circulatory and renal failure than patients with non-emm1/T1 (p = 0.036 and p = 0.007, respectively). After correction for severity of illness (SAPS 3) and age, iGAS infection was associated with lower mortality risk (95% confidence interval (CI) of hazard ratio (HR) 0.204-0.746, p <  0.001). Morbidity analyses demonstrated that iGAS patients were more likely to develop renal failure. CONCLUSION: Critically ill patients with iGAS infection had a lower mortality risk but a higher degree of renal failure compared to similarly ill sepsis patients. emm1/T1 was found to be the most dominant serotype, and patients with emm1/T1 demonstrated more circulatory and renal failure than patients with other serotypes of iGAS.

The majority of MRSA colonized children not given eradication treatment are still colonized one year later. Systemic antibiotics improve the eradication rate.

BACKGROUND: Colonization with methicillin-resistant Staphylococcus aureus (MRSA) can cause endogenously derived infections and be a source of transmission to other people. Neither colonization time of asymptomatic MRSA colonization nor the effect of treatment aiming at MRSA eradication in children has been thoroughly investigated. METHODS: Two hundred ninety-three children <18 years in the mandatory follow-up program for MRSA-carriers in Malmö, Sweden were evaluated. Samples from the throat, nares, perineum and skin lesions from each child were screened for MRSA with a PCR-based broth enrichment method. PVL presence and spa-type were evaluated in a majority of cases. The sampling was repeated approximately every 6 month after initial detection. When three consecutive sets of negative samples during at least a 6-month period were obtained, the MRSA was considered permanently eradicated. MRSA eradication treatment given, on clinical grounds during follow-up, was noted. RESULTS: One year after detection 62% of the untreated children were still MRSA positive and after 2 years 28%. MRSA throat colonization and having MRSA positive household contacts significantly prolonged the observed colonization time. Topical MRSA eradication treatment was successful in 36% of cases and in 65% if systemic antibiotics were added. Presence of PVL correlated with shorter observed colonization time in the older age group and with increased eradication success among throat carriers. CONCLUSION: MRSA throat colonization and having MRSA positive household contacts prolongs the time of MRSA colonization in children. Systemic antibiotics enhance the effect of MRSA eradication treatment.

Molecular detection of Borrelia burgdorferi sensu lato - An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories.

INTRODUCTION: Lyme borreliosis (LB) is the most common tick transmitted disease in Europe. The diagnosis of LB today is based on the patient´s medical history, clinical presentation and laboratory findings. The laboratory diagnostics are mainly based on antibody detection, but in certain conditions molecular detection by polymerase chain reaction (PCR) may serve as a complement. AIM: The purpose of this study was to evaluate the analytical sensitivity, analytical specificity and concordance of eight different real-time PCR methods at five laboratories in Sweden, Norway and Denmark. METHOD: Each participating laboratory was asked to analyse three different sets of samples (reference panels; all blinded) i) cDNA extracted and transcribed from water spiked with cultured Borrelia strains, ii) cerebrospinal fluid spiked with cultured Borrelia strains, and iii) DNA dilution series extracted from cultured Borrelia and relapsing fever strains. The results and the method descriptions of each laboratory were systematically evaluated. RESULTS AND CONCLUSIONS: The analytical sensitivities and the concordance between the eight protocols were in general high. The concordance was especially high between the protocols using 16S rRNA as the target gene, however, this concordance was mainly related to cDNA as the type of template. When comparing cDNA and DNA as the type of template the analytical sensitivity was in general higher for the protocols using DNA as template regardless of the use of target gene. The analytical specificity for all eight protocols was high. However, some protocols were not able to detect Borrelia spielmanii, Borrelia lusitaniae or Borrelia japonica.

Clinical and microbiological features of bacteremia with Streptococcus equi.

Streptococcus equi (SE) rarely causes human infections. We identified 18 SE isolates from blood cultures. The focus of infection was unknown (n = 5), arthritis (n = 3), catheter-related (n = 2), pneumonia (n = 2), or other (n = 6). There were no fatalities. Several patients had animal contacts but there were no indications of clonal outbreaks.

Quantitative proteogenomics of human pathogens using DIA-MS.

The increasing number of bacterial genomes in combination with reproducible quantitative proteome measurements provides new opportunities to explore how genetic differences modulate proteome composition and virulence. It is challenging to combine genome and proteome data as the underlying genome influences the proteome. We present a strategy to facilitate the integration of genome data from several genetically similar bacterial strains with data-independent analysis mass spectrometry (DIA-MS) for rapid interrogation of the combined data sets. The strategy relies on the construction of a composite genome combining all genetic data in a compact format, which can accommodate the fusion with quantitative peptide and protein information determined via DIA-MS. We demonstrate the method by combining data sets from whole genome sequencing, shotgun MS and DIA-MS from 34 clinical isolates of Streptococcus pyogenes. The data structure allows for fast exploration of the data showing that undetected proteins are on average more amenable to amino acid substitution than expressed proteins. We identified several significantly differentially expressed proteins between invasive and non-invasive strains. The work underlines how integration of whole genome sequencing with accurately quantified proteomes can further advance the interpretation of the relationship between genomes, proteomes and virulence. This article is part of a Special Issue entitled: Computational Proteomics.

Epidemiology and antibiotic susceptibility of aerococci in urinary cultures.

In this study, we present population-based data regarding the prevalence of aerococci in clinical urinary samples. During a 3-month period, all aerococcal isolates from urinary samples from 2 clinical microbiology laboratories were collected. We identified 64 Aerococcus urinae isolates and 40 Aerococcus sanguinicola isolates, which correlates with an incidence of 33 cases of aerococcal bacteriuria per 100,000 inhabitants per year. The median age was 83years for all patients with aerococcal bacteriuria, which was significantly higher than for patients with Escherichia coli or Enterococcus faecalis bacteriuria. Sex was almost equally distributed between men and women with aerococcal bacteriuria, whereas females dominated in E. coli bacteriuria. The aerococcal isolates displayed low MICs for ampicillin, cefalotin, mecillinam, and nitrofurantoin. Most A. sanguinicola isolates were resistant to ciprofloxacin, whereas most A. urinae isolates had low MICs. Clinical studies are needed to establish clinical breakpoints and optimal treatment.

Midkine is expressed and differentially processed during chronic obstructive pulmonary disease exacerbations and ventilator-associated pneumonia associated with Staphylococcus aureus infection.

Staphylococcus aureus is sometimes isolated from the airways during acute exacerbations of chronic obstructive pulmonary disease (COPD) but more commonly recognized as a cause of ventilator-associated pneumonia (VAP). Antimicrobial proteins, among them midkine (MK), are an important part of innate immunity in the airways. In this study, the levels and possible processing of MK in relation to S. aureus infection of the airways were investigated, comparing COPD and VAP, thus comparing a state of disease with preceding chronic inflammation and remodeling (COPD) with acute inflammation (that is, VAP). MK was detected in the small airways and alveoli of COPD lung tissue but less so in normal lung tissue. MK at below micromolar concentrations killed S. aureus in vitro. Proteolytic processing of MK by the staphylococcal metalloprotease aureolysin (AL), but not cysteine protease staphopain A (SA), resulted in impaired bactericidal activity. Degradation was seen foremost in the COOH-terminal portion of the molecule that harbors high bactericidal activity. In addition, MK was detected in sputum from patients suffering from VAP caused by S. aureus but less so in sputum from COPD exacerbations associated with the same bacterium. Recombinant MK was degraded more rapidly in sputum from the COPD patients than from the VAP patients and a greater proteolytic activity in COPD sputum was confirmed by zymography. Taken together, proteases of both bacteria and the host contribute to degradation of the antibacterial protein MK, resulting in an impaired defense of the airways, in particular, in COPD where the state of chronic inflammation could be of importance.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry is a sensitive and specific method for identification of aerococci.

Conventional methods for the identification of human-pathogenic aerococci to the species level are not reliable. We show that matrix-assisted laser desorption ionization-time of flight mass spectrometry correctly identifies aerococci to the species level and that it can be used to identify aerococci with high specificity in the diagnostic clinical microbiology laboratory.

Spontaneously draining acute otitis media in children: an observational study of clinical findings, microbiology and clinical course.

OBJECTIVES: To study the outcome of acute otitis media (AOM) with otorrhoea in children managed initially without antibiotics, in relation to bacterial and clinical findings, and to identify those who may benefit from antibiotics. METHODS: Otherwise healthy, not otitis prone children aged 2-16 y, presenting with AOM with spontaneous otorrhoea, were recruited from primary care and followed at selected ear, nose and throat (ENT) clinics. Specimens for bacterial investigations were obtained; symptoms were registered on a daily basis. The main outcomes measured were the frequency of children treated with antibiotics due to persisting AOM within 9 days in relation to clinical and bacteriological findings, and new AOM within 3 months. RESULTS: Twelve of 71 children who completed the trial received antibiotics during the first 9 days due to lack of improvement. One received antibiotics after 16 days due to relapsing AOM and 6 received antibiotics after 30 days due to new AOM. At 2-4 days following inclusion, over 70% of children showed normalized eardrum status and markedly reduced secretion. Alloiococcus otitidis was found in 23 samples, Streptococcus pneumoniae in 12, Streptococcus pyogenes in 6, and Fusobacterium nucleatum in 5. Mycoplasma pneumoniae, Chlamydia pneumoniae, and Fusobacterium necrophorum were not detected. Antibiotics were prescribed more extensively to children with a pulsating eardrum and abundant purulent secretion. All children with S. pyogenes received antibiotics, whereas children with only A. otitidis did not. CONCLUSIONS: The results suggest that antibiotics are indicated in AOM with otorrhoea and the presence of abundant purulent secretion, a pulsating eardrum, or the presence of S. pyogenes. The presence of only A. otitidis was not associated with a more prolonged course or the need for antibiotics.

Pseudomonas aeruginosa in cystic fibrosis: pyocyanin negative strains are associated with BPI-ANCA and progressive lung disease.

The clinical consequence of chronic Pseudomonas aeruginosa colonization in cystic fibrosis (CF) varies between individuals for unknown reasons. Auto-antibodies against bactericidal/permeability increasing protein (BPI-ANCA) are associated with poor prognosis in CF. We hypothesize that there is a correlation between the presence of BPI-ANCA, the properties of the colonizing bacteria and the clinical conditions of the host. We compared isolates of P. aeruginosa from BPI-ANCA positive CF patients who have deteriorating lung disease with BPI-ANCA negative CF patients who are in stable clinical conditions. Epithelial cells (A549) and isolated polymorphonuclear granulocytes (PMNs) were stimulated with the isolates and cell death was analyzed with flow cytometry. We found that the ANCA associated strains in most cases showed pyocyanin negative phenotypes. These strains also induced less inflammatory response than the non-ANCA associated strains as shown by apoptosis and necrosis of epithelial cells and neutrophils. Our results suggest that colonization with strains of P. aeruginosa that induce a weak inflammatory response is associated with unfavorable outcome in CF. We speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying PMNs in the airways, contributing to progression in CF lung disease.

Axillary abscess complicated by venous thrombosis: identification of Streptococcus pyogenes by 16S PCR.

We report a case of an axillary abscess with Streptococcus pyogenes complicated by venous thrombosis. Bacterial etiology and typing were obtained by PCR and sequencing of the 16S rRNA and M-protein genes from abscess material. The bacterium was of serotype M41, and serology indicated that it had expressed procoagulant factors.