Diagnosis and therapy of Mycobacterium marinum: a single-center 21-year retrospective analysis.

BACKGROUND AND OBJECTIVES: In Europe, infections with Mycobacterium (M.) marinum are rare. We conducted a retrospective single-center study to assess the clinical spectrum of M. marinum infection and its diagnosis, treatment and outcome under real-world conditions. PATIENTS AND METHODS: Eighteen patients presenting with M. marinum infections between 1998 and 2018 were identified in the data warehouse of the University Hospital Würzburg and considered for detailed analysis. RESULTS: Twelve patients reported aquatic exposure. In 16/18 cases the upper extremities were affected. No invasive infections were detected. Mean time to diagnosis was 15 weeks. Histology revealed granulomatous inflammation in 14 patients while mycobacterial cultures were positive for M. marinum in 16 cases. Most patients received antibiotic monotherapy (14/18) while combination therapy was administered in four cases. Treatment (with a median duration of 10 weeks) was successful in 13 patients. Five patients were lost to follow-up. CONCLUSIONS: Our retrospective analysis of M. marinum infections at a German tertiary referral center revealed a considerable diagnostic delay and the relevance of microbiological culture, PCR and histology for diagnosis. Monotherapy with clarithromycin (rather than doxycycline) appeared as a reasonable treatment option while immunosuppressed or -compromised patients and those with extended disease received combination therapy.

Performance of Three SARS-CoV-2 Immunoassays, Three Rapid Lateral Flow Tests, and a Novel Bead-Based Affinity Surrogate Test for the Detection of SARS-CoV-2 Antibodies in Human Serum.

For the control of immunity in COVID-19 survivors and vaccinated subjects, there is an urgent need for reliable and rapid serological assays. Based on samples from 63 COVID-19 survivors up to 7 months after symptom onset, and on 50 serum samples taken before the beginning of the pandemic, we compared the performances of three commercial immunoassays for the detection of SARS-CoV-2 IgA and IgG antibodies (Euroimmun SARS-COV-2 IgA/IgG, Mikrogen recomWell SARS-CoV-2 IgA/IgG, and Serion ELISA agile SARS-CoV-2 IgA/IgG) and three rapid lateral flow (immunochromatographic) tests (Abbott PanBio COVID-19 IgG/IgM, Nadal COVID-19 IgG/IgM, and Cleartest Corona 2019-nCOV IgG/IgM) with a 50% plaque-reduction neutralization test (PRNT50) representing the gold standard. Fifty-seven out of 63 PCR-confirmed COVID-19 patients (90%) showed neutralizing antibodies. The sensitivity of the seven assays ranged from 7.0% to 98.3%, and the specificity ranged from 86.0% to 100.0%. Only one commercial immunoassay showed a sensitivity and specificity of greater than 98%.

Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis.

CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5' ends formed by transcription and not by processing. Although crRNA 3' end formation involves RNase III and trans-encoded tracrRNA, as in other type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/Cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.

The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

Neisseria meningitidis, a commensal ß-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.

The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq.

Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of -35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.

Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence.

BACKGROUND: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. RESULTS: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. CONCLUSIONS: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.

Co-infections in Visceral Pentastomiasis, Democratic Republic of the Congo.

Snakeborne Armillifer pentastomiasis is an emerging human parasitic infection in rural tropical areas where snake meat is eaten. After a series of severe ocular A. grandis larval infections and anecdotal abdominal infection in Sankuru District, Democratic Republic of the Congo, during 2014-2015, we systematically investigated possible pentastomid etiology in patients who underwent surgery in the region. Histologic and molecular analyses by established pentastomid 18S rDNA- and newly developed Armillifer-specific cytochrome oxidase PCRs revealed larval pentastomid lesions in 3.7% of patients. Some persons had A. armillatus and A. grandis co-infections. Another pentastomid larva, Raillietiella sp., was molecularly detected in 1 patient who had concomitant A. grandis and A. armillatus infection. The PCRs used were suitable for detecting pentastomid species even in highly necrotic tissues. Phylogenetic analyses of Armillifer cytochrome oxidase genes detected multiple local strains.

Searching for success: Development of a combined patient-reported-outcome ("PRO") criterion for operationalizing success in multi-modal pain therapy.

BACKGROUND: There is a need for a way to measure success in multi-modal pain therapy that researchers and clinicians can agree upon. According to developments in health services research, operationalizing success should take patient-reported outcomes into account. We will present a success criterion for pain therapy that combines different patient-reported variables and includes validity measures. The usable criterion should be part of a statistically significant and satisfactory model identifying predictors of successful pain therapy. METHODS: Routine data from 375 patients treated with multi-modal pain therapy from 2008 to 2013 were used. The change scores of five constructs were used for the combined success criterion: pain severity, disability due to pain, depressiveness, and physical- and mental-health-related quality of life. According to the literature, an improvement of at least ½ standard deviation was required on at least four of the five constructs to count as successful. A three-step analytical approach including multiple binary logistic regression analysis was chosen to identify the predictors of therapy success with the success criterion as the dependent variable. RESULTS: A total of 58.1% of the patients were classified as successful. Convergent and predictive validity data show significant correlations between the criterion and established instruments, while discriminative validity could also be shown. A multiple binary logistic regression analysis confirmed the feasibility; a significant model (Chi(2) (8) = 52.585; p < .001) that explained 17.6% of the variance identified the following predictors of therapy success: highest pain severity in the last 4 weeks, disability due to pain, and number of physician visits in the last 6 months. CONCLUSIONS: It is possible to develop a feasible success criterion that combines several variables and includes patient-reported outcomes ("PROs") with routine data that can be used in a predictor analysis in multi-modal pain therapy. The criterion was based on basic constructs used in pain therapy and used widespread validated self-rating instruments. Thus, it should be easy to transfer this criterion to other institutions.

Empiric Antibiotic Therapy in 1402 Children With Parapneumonic Effusion/Pleural Empyema in Germany: A Long-term Surveillance Study.

OBJECTIVE: This study investigated empiric antibiotic treatment (EAT), guideline adherence, antibiotic streamlining and clinical outcomes in 1402 hospitalized children with pediatric parapneumonic effusion/pleural empyema (PPE/PE). METHODS: A nationwide surveillance study collected data on EAT, clinical course/outcome, pathogens, susceptibility testing and antibiotic streamlining of children with PPE/PE in Germany between 2010 and 2018. Subgroups were compared using χ2 test/Fisher exact test, Mann-Whitney U test and linear regression analysis adjusting for patient age where appropriate. RESULTS: Complete data on EAT were available for 1402 children. In children with monotherapy (n = 567) and in children with combination therapy of 2 antibiotics (n = 589), the most commonly used antibiotics were aminopenicillin/beta-lactamase inhibitor [138/567 (24.3%) and 102/589 (17.3%)] and cefuroxime [291/567 (51.3%) and 294/589 (49.9%)]. The most common combinations with these beta-lactams were macrolides, aminoglycosides and clindamycin. We observed no difference in clinical severity/outcome between EAT with aminopenicillin/beta-lactamase inhibitor and cefuroxime, neither when used in monotherapy nor when used in combination therapy of 2 antibiotics. Species diagnosis of Streptococcus pneumoniae (n = 192), Streptococcus pyogenes (n = 111) or Staphylococcus aureus (n = 38) in polymerase chain reaction or culture from pleural fluid or blood resulted in a switch to an appropriate narrow-spectrum beta-lactam therapy in 9.4%, 18.9 % and 5.2% of children. In a subset of children with reported bacterial susceptibility testing, penicillin resistance was reported in 3/63 (4.8%) of S. pneumoniae and methicillin resistance in S. aureus was reported in 10/32 (31.3%) of children. CONCLUSION: This study points to antibiotic overtreatment in children with PPE/PE, particularly the frequent use of combinations of antibiotics. Children receiving combinations of antibiotics did not show differences in clinical outcomes. The low rate of children with streamlined antibiotic therapy even upon pathogen detection indicates a necessity for antibiotic stewardship measures in PPE/PE and the need of investigating other potential therapeutic strategies as anti-inflammatory therapy.

The zinc-responsive regulon of Neisseria meningitidis comprises 17 genes under control of a Zur element.

Zinc is a bivalent cation essential for bacterial growth and metabolism. The human pathogen Neisseria meningitidis expresses a homologue of the Zinc uptake regulator Zur, which has been postulated to repress the putative zinc uptake protein ZnuD. In this study, we elucidated the transcriptome of meningococci in response to zinc by microarrays and quantitative real-time PCR (qRT-PCR). We identified 15 genes that were repressed and two genes that were activated upon zinc addition. All transcription units (genes and operons) harbored a putative Zur binding motif in their promoter regions. A meningococcal Zur binding consensus motif (Zur box) was deduced in silico, which harbors a conserved central palindrome consisting of hexameric inverted repeats separated by three nucleotides (TGTTATDNHATAACA). In vitro binding of recombinant meningococcal Zur to this Zur box was shown for the first time using electrophoretic mobility shift assays. Zur binding to DNA depended specifically on the presence of zinc and was sensitive to mutations in the palindromic sequence. The Zur regulon among genes of unknown function comprised genes involved in zinc uptake, tRNA modification, and ribosomal assembly. In summary, this is the first study of the transcriptional response to zinc in meningococci.

Genome sequence of a Neisseria meningitidis capsule null locus strain from the clonal complex of sequence type 198.

Neisseria meningitidis is a commensal and accidental pathogen exclusively of humans. Although the production of polysaccharide capsules is considered to be essential for meningococcal virulence, there have been reports of constitutively unencapsulated strains causing invasive meningococcal disease (IMD). Here we report the genome sequence of a capsule null locus (cnl) strain of sequence type 198 (ST-198), which is found in half of the reported cases of IMD caused by cnl meningococcal strains.

Molecular detection of Campylobacter jejuni as a cause of culture-negative spondylodiscitis.

Spondylodiscitis caused by Campylobacter species is a rare disease which is most often caused by Campylobacter fetus. We report a case of culture-negative spondylodiscitis and a psoas abscess due to Campylobacter jejuni in a 68-year-old woman, as revealed by 16S rRNA gene and Campylobacter-specific PCRs from biopsied tissue.

Periprosthetic joint infection: Comparison of automated multiplex-PCR Unyvero i60 ITI cartridge system with bacterial culture and real-time PCR.

BACKGROUND: In the past, various efforts have been made to investigate diagnostic tools for periprosthetic-joint-infection (PJI). It is little-known about the diagnostic utility of polymerase-chain-reaction (PCR) in this context, especially concerning the role of multiplex-PCR assays comparing with conventional tissue culture. OBJECTIVE: Evaluation of an automated-multiplex-PCR cartridge system for patients with suspicion of PJI in comparison with conventional microbiological culture and 16S-rDNA-PCR. METHODS: On suspicion of PJI synovial fluid specimen were taken preoperatively or periprosthetic tissue was collected intraoperatively. Microbiological analysis included conventional culture, 16S-rDNA-PCR and automated-multiplex-PCR (Unyvero-i60-ITI®). The European-Bone-and-Joint-Infection-Society (EBJIS) criteria were used for PJI diagnosis. Positive and negative percent agreement was calculated. Total percentage agreement and Cohen's kappa coefficient were calculated. Sensitivity, specificity and positive predictive value of conventional culture, 16S-rDNA-PCR and multiplex-PCR were calculated. Ten specimens of proved PJI were used as control group. RESULTS: Fifty specimen were suitable for culture. 14 (28%) were classified as PJI, 36 (72%) were aseptic. Coagulase-negative staphylococci was the most frequent detected pathogen. Concordance-rate between mPCR and culture results was 75.6% with a Cohen's kappa of 0.28. Concordance-rate between mPCR and 16S-rDNA was 82.9%, Cohen's kappa was 0.13. Concordance analysis between culture results and 16S-rDNA lead to a concordance-rate of 88.9%. Cohen's kappa was calculated with 0.6. With regard to the microbiological culture as reference, sensitivity of the mPCR was 0.33 and specificity was 0.91. Sensitivity and specificity of the 16S-rDNA-PCR was 0.55 and 0.97. The positive predictive value was 0.57 for the mPCR and 0.83 for the 16S-rDNA-PCR. CONCLUSIONS: Due to fair agreement between mPCR and conventional microbiological culture, the tested multiplex-PCR could be an additional instrument for the detection of PJI but is not superior over the conventional culture.

A streamlined method for the fast and cost-effective detection of bacterial pathogens from positive blood cultures for the BacT/ALERT blood culture system using the Vitek MS mass spectrometer.

BACKGROUND AND OBJECTIVE: Prompt pathogen identification of blood stream infections is essential to provide appropriate antibiotic treatment. Therefore, the objective of this prospective single centre study was to establish an inexpensive, fast and accurate protocol for bacterial species identification with SDS protein-extraction directly from BacT/Alert® blood culture (BC) bottles by VitekMS®. RESULTS: Correct species identification was obtained for 198/266 (74.4%, 95%-CI = [68.8%, 79.6%]) of pathogens. The protocol was more successful in identifying 87/96 (91.4%, 95%-CI = [83.8%, 93.2%]) gram-negative bacteria than 110/167 (65.9%, 95%-CI = [58.1%, 73.0%]) gram-positive bacteria. The hands-on time for sample preparation and measurement was about 15 min for up to five samples. This is shorter than for most other protocols using a similar lysis-centrifugation approach for the combination of BacT/Alert® BC bottles and the Vitek® MS mass spectrometer. The estimated costs per sample were approx. 1.80€ which is much cheaper than for commercial kits. CONCLUSION: This optimized protocol allows for accurate identification of bacteria directly from blood culture bottles for laboratories equipped with BacT/Alert® blood culture bottles and VitekMS® mass spectrometer.

Farming Practice Influences Antimicrobial Resistance Burden of Non-Aureus Staphylococci in Pig Husbandries.

Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.

Whole-genome sequence of the transformable Neisseria meningitidis serogroup A strain WUE2594.

Serogroup A meningococci are a leading cause of bacterial meningitis in children and young adults worldwide. However, the genetic basis of serogroup A strains' virulence and their epidemiological properties remain poorly understood. Therefore, we sequenced the complete genome of the transformable Neisseria meningitidis serogroup A strain WUE2594.

Transmission of Armillifer armillatus ova at snake farm, The Gambia, West Africa.

Visceral pentastomiasis caused by Armillifer armillatus larvae was diagnosed in 2 dogs in The Gambia. Parasites were subjected to PCR; phylogenetic analysis confirmed relatedness with branchiurans/crustaceans. Our investigation highlights transmission of infective A. armillatus ova to dogs and, by serologic evidence, also to 1 human, demonstrating a public health concern.

Characterization of FarR as a highly specialized, growth phase-dependent transcriptional regulator in Neisseria meningitidis.

Transcriptional regulators play an important role for the survival of Neisseria meningitidis within its human host. We have recently shown that FarR acts as transcriptional repressor of the adhesin nadA in N. meningitidis. Here, we examined the FarR regulon by microarray analyses, qRT-PCR, and electrophoretic mobility shift assays, revealing that FarR is a highly specific repressor of nadA. We demonstrate by reporter gene fusion assays that alterations of the FarR binding site within the nadA promoter are sufficient to induce transcription of nadA. Furthermore, farR expression is growth phase-dependent. The highest transcription rate was observed in the late-exponential growth phase of meningococci. Upon contact with active components of the complement system in normal human serum, expression of farR is slightly downregulated. Concluding, we present FarR as an exquisitely specialized, growth phase-dependent, possibly complement-responsive transcriptional regulator in N. meningitidis.

Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines.

BACKGROUND: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. RESULTS: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptor-mediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. CONCLUSIONS: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.