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The monitoring of antimicrobial resistance is becoming a priority mainly in developing countries in order to control the emergence of multi-resistant bacteria strains. This study evaluated the antibiotic resistance profile of bacteria isolated at the National Institute of Hygiene in Togo. Lab records from January 2013 to December 2015 were examined. A total of 33,147 samples were analyzed. Among them, vaginal swabs and urines were more represented with 38.17% and 33.24% respectively. 3,497 Gram-positive and 1,970 Gram-negative bacterial strains were cultured. Enterobacteriaceae (57.6%), Staphylococcus spp. (21.1%) and Streptococcus spp (10.5%) were more isolated. Escherichia coli was resistant over the three years to piperacillin (79.66%, 75.1%, 83%), trimethoprim/sulfamethoxazole (79.3%, 82%, 82.8%), ticarcillin (89%, 89.7%, 93%) and amoxicillin (94.1%, 94%, 96.09%); whereas Staphylococcus aureus was resistant to penicillin G. Streptococcaceae isolates was resistant to trimethoprim/sulfamethoxazole (78.11-87.1%), tetracycline (82.2-91.16%) and norfloxacin (86.16-94.3%). Escherichia coli and Staphylococcus aureus isolated from urines were more resistant to antibiotics than those isolated from vaginal swabs. There is a need to develop new strategies in order to fight against the emergence of multi-resistant bacteria in Togo.

Sepsis, a life-threatening organ dysfunction, continues to cause considerable morbidity. Reliable animal models to evaluate therapeutic options are urgently required, however, some of the most esta...

BACKGROUNDSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (COVID-19) have afflicted millions of people in a worldwide pandemic. Several vaccines have been developed to prevent infection and illness. However, the safety and efficacy of most of the vaccines currently available are still being questioned by part of the public opinion. Even if vaccine-resistant individuals represent a minority in most countries, their hesitancy is sufficient to delay the highly desired ‘herd-immunity threshold.’ METHODSIn an ongoing multinational trial, we collected blood samples from 365 adults, 18 years of age or older, vaccinated with mRNA vaccines (Moderna, BioNTech), viral DNA-vectored vaccines (AstraZeneca, Sputnik-V, and Johnson and Johnson), or the attenuated virus vaccine from Sinopharm. Out of the 365 vaccinated individuals included in the study, 41 received two doses of Moderna Biotech's Spikevax, 92 received two doses of BioNTech’s Comirnaty, 52 were vaccinated with two doses of Oxford-AstraZeneca’s Vaxzevria, 34 received one dose of Johnson and Johnson’s Jansen, 35 two doses of Gamaleja’s Sputnik-V and 28 two doses of Sinopharm’s BBIBP-CorV. In addition, 40 received a prime dose of AstraZeneca followed by BioNTech as a booster, whereas 43 received Moderna’s vaccine as a booster after a prime dose of AstraZeneca. After collecting reactogenicity data, the expression of S-Protein binding IgG and IgA were analyzed before and after full vaccination in each group using an automated sandwich ELISA system. In addition, the neutralizing capacity of sera from individuals from all groups was investigated using an ACE-2-RBD neutralizing assay. RESULTSThe main side effects reported included short-term mild-to-moderate pain at the injection site, fatigue, and headache. More severe side effects were reported by vaccinees in the Moderna (10%), AstraZeneca (11%), Johnson and Johnson (5.9%), and Sputnik-V (7.2%) groups. No severe adverse reaction was reported in the BioNTech group, and the Sinopharm vaccinees presented the mildest reactogenicity profile, with 93.8% of the vaccinees declaring no adverse reactions. Moderna’s vaccine induced the highest amounts of SARS-CoV-2 specific IgG, IgA, and serum neutralization activity compared to the other groups. In contrast, people vaccinated with Sinopharm and Johnson and Johnson’s vaccines have the lowest SARS-CoV-2-specific antibody titers. Vaccinees from the Johnson and Johnson group presented significant levels of SARS-CoV-2 specific IgA but not IgG compared to the controls before vaccination. In the Sinopharm group, neither IgG nor IgA expression was significant. In addition, sera from vaccinees of these two groups presented no significant neutralization potential compared to the unvaccinated controls. Significant negative correlations between age and SARS-CoV-2- specific IgG expression were observed in the Johnson and Johnson (r=-0.4414, p=0.009) and Sinopharm (r=-0.6108, p=0.0006) groups. Remarkably, younger vaccinees (18-60 years old) in both Sinopharm and Johnson and Johnson groups produced substantial SARS-CoV-2 specific antibody expression and exhibited significant neutralization potential. While the AstraZeneca vaccine alone induced moderate IgG and IgA expression, the combination with Moderna or BioNTech mRNA vaccines induced higher antibody levels than a double dose of AstraZeneca and similar IgG expression and neutralization potential compared to Moderna, or BioNTech used alone. CONCLUSIONThe results suggest that the Moderna vaccine is the most immunogenic after two doses. AstraZeneca and Sputnik-V presented moderate but significant antibody expression and virus neutralizing properties. Low antibody and neutralization potential was observed in the elderly vaccinated with Sinopharm or Johnson and Johnson vaccines. The data also suggest that heterologous vaccination strategies combining the AstraZeneca DNA vectored vaccines and mRNA vaccines Moderna or BioNTech booster induced more robust antibody and virus neutralization potential compared to their homologous counterparts.

Background Contrary to the predictions, prevalence and mortality due to COVID-19 have remained moderate on the African continent. Several factors, including age, genetics, vaccines, and co-infections, might impact the course of the pandemic in Africa. Helminths are highly endemic in Sub-Saharan Africa and are renowned for their ability to modulate their host immune reactions. Methods Here we analyzed in vitro the impact of major helminth antigens on the immune reactivity to SARS-CoV-2 in COVID-19 patients using flow cytometry and Luminex. Results: We observed that helminth antigens significantly reduced the frequency of SARS-CoV-2-reactive CD4+ T helper cells. In contrast, the expression of SARS-CoV-2-reactive CD8+ T cells was not affected. In addition, stimulation with helminth antigens was associated with increased IL-10 and a reduction of IFNγ and TNFα. Conclusion: Our data offer a plausible explanation for the moderate incidence of COVID-19 in Africa and support the hypothesis that helper T cell-mediated immune responses to SARS-CoV-2 are mitigated in the presence of helminth antigens, while virus-specific cytotoxic T cell responses are maintained.

The coronavirus disease 2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2, a recently discovered strain of coronavirus. The virus has spread rapidly, causing millions of death worldwide. Contrary to the predictions, prevalence and mortality due to COVID-19 have remained moderate on the African continent. Several factors, including age, genetics, vaccines, and co-infections, might impact the course of the pandemic in Africa. Helminths are highly endemic in Sub-Saharan Africa and are renowned for their ability to evade, skew, and suppress human immune responses through various immune-modulatory mechanisms. Such effects will likely impact SARS-CoV-2 transmission and disease progression.Here, we analyzed in vitro the impact of antigen extracts from three major helminth parasites, including Onchocerca volvulus, Brugia malayi, and Ascaris lumbricoides, on the immune reactivity to SARS-CoV-2 peptides in COVID-19 patients. Activation of CD4We observed that helminth antigens significantly reduced the frequency of SARS-CoV-2-reactive CD4Our data offer a plausible explanation for the moderate incidence of COVID-19 in Africa and support the hypothesis that helper T cell-mediated immune responses to SARS-CoV-2 are mitigated in the presence of helminth antigens, while virus-specific cytotoxic T cell responses are maintained.

Antibiotic resistance represents a major global concern. The rapid spread of opportunistically pathogenic carbapenemase-encoding bacteria (CEB) requires clinicians, researchers, and policy-makers to swiftly find solutions to reduce transmission rates and the associated health burden. Epidemiological data is key to planning control measures. Our study aims to contribute by providing an analysis of 397 unique CEB isolates detected in a tertiary hospital in Germany. We propose new findings on demographic variables to support preventive sanitary precautions in routine clinical practice. Data on detected CEB was combined with patient’s demographic and clinical information for each isolate. Multiple regression techniques were applied to estimate the predictive quality of observed differences. Our findings confirm the role of age and gender in CEB colonization patterns and indicate a role for ethnicity and domicile. Also, carbapenemase-encoding A. baumannii was most frequently introduced to the hospital, while the risk of colonization with VIM-encoding P. aeruginosa rose with the length of hospital stay. P. aeruginosa remains an important complication of prolonged hospital stays. The strong link to hospital-wastewater may have implications for hospital-built environments. A. baumannii can be efficiently controlled from spreading at hospital admission. OXA-encoding CEB being harder to detect in routine screening, targeted preventive measures, such as culture media selective for carbapenem-resistant bacteria, would be opportune for patients from selected regions. The CEB differences linked to ethnicity found in our study may further be supporting the tailoring of diagnostic approaches, as well as health policies upon confirmation by other studies and a better understanding of their global distribution.

In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l−1 and MICs of cefotaxime ranged from 128 mg l−1 whereas MICs of NIT ranged from 0.125 to 2 mg l−1 . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.

Recreational use of bathing waters is very popular in Germany and the European Union (EU). Therefore, the spread of antibiotic-resistant facultative-pathogenic bacteria (ARB) presents a possible public health issue. During the 2018 summer bathing season, a prevalence study involving 16 selected EU bathing waters in North Rhine-Westphalia was conducted. The aim was to identify the presence of ARB, antibiotic residues or resistance genes, and to develop a tool that assists advisers on prevention measures for individuals and the health authorities. The study included (i) hygienic-microbiological analyses according to the EU Bathing Water Directive, (ii) antibiotic residue measurements, (iii) cultural and molecular analyses to identify ARB and resistance genes and (iv) development of a risk assessment tool based on the burden of the bathing water and the vulnerability of bathing persons. All bathing waters were complied with the EU Bathing Water Directive. Three samples were found to contain very small amounts of ARB. Small quantities of antibiotic residues were detected in three bathing waters. Only one sample contained a resistance gene. In total, only 2 bathing waters were assumed to be systemically burdened with ARB and antibiotic residues or resistance genes. 4 bathing waters indicated single contamination events, 10 bathing waters were unburdened. In conclusion, bathing waters fulfilling criteria of the EU bathing water directive did not show high contaminations with ARB, antibiotic residues or resistance genes. However, we propose that people with risk factors should avoid swimming in burdened waters; people with immune deficiency should avoid swimming at all.

Berichtet wird uber den bemerkenswerten Verlauf eines fazialen Ulkus bei einer Patientin unter Therapie mit Prednisolon im Rahmen eines M. Crohn. Bei initial unklarer Diagnose zeigte sich ein rasch progredienter Befund trotz dreifach antiinfektiver Therapie (antiviral, antibiotisch, antimykotisch). Eine Infektion mit Orthopoxviren konnte anhand umfangreicher Diagnostik im Verlauf bestatigt werden, relevante Differentialdiagnosen wurden ausgeschlossen. Nach umfangreicher nekrotischer Umwandlung kam es nach Monaten zu einer narbigen Abheilung. Kuhpockeninfektionen kommen in Deutschland immer wieder vor und sind eine klinisch relevante Zoonose. Am haufigsten erfolgt die Ubertragung durch Katzen und Nagetiere. Differenzialdiagnostisch kommen verschiedenste teilweise seltene Erreger ursachlich in Betracht, die hier anhand klinischer und histopathologischer Merkmale diskutiert werden. Hierzu zahlen bakterielle, mykobakterielle, mykotische und parasitare Infektionen. Insbesondere die kutane Leishmaniose ist aufgrund steigender Inzidenz hierzulande explizit zu nennen. Da infektiose faziale Ulzera bei inadaquater Therapie zu lebensgefahrlichen Komplikationen und ausgedehnter entstellender Narbenbildung fuhren konnen, ist eine zeitkritische kalkulierte Therapieeinleitung vordringlich.

This study assessed the diagnostic performance of the new coronavirus disease 2019 (COVID-19) SEROSpeed IgM/IgG Rapid Test (BioSpeedia, a spinoff of the Pasteur Institute of Paris) for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in comparison to other commercial antibody assays through a large cross-European investigation. The clinical specificity was assessed on 215 prepandemic sera (including some from patients with viral infections or autoimmune disorders)., This study assessed the diagnostic performance of the new COVID19SEROSpeed IgM/IgG rapid test (BioSpeedia, a spinoff of the Pasteur Institute of Paris) for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in comparison to other commercial antibody assays through a large cross-European investigation. The clinical specificity was assessed on 215 prepandemic sera (including some from patients with viral infections or autoimmune disorders). The clinical sensitivity was evaluated on 710 sera from 564 patients whose SARS-CoV-2 infection was confirmed by quantitative reverse transcription-PCR (qRT-PCR) and whose antibody response was compared to that measured by five other commercial tests. The kinetics of the antibody response were also analyzed in seven symptomatic patients. The specificity of the test (BioS) on prepandemic specimens was 98.1% (95% confidence interval [CI], 96.2% to 99.4%). When tested on the 710 pandemic specimens, BioS showed an overall clinical sensitivity of 86.0% (95% CI, 0.83 to 0.89), with good concordance with the Euroimmun assay (overall concordance of 0.91; Cohen’s kappa coefficient of 0.62). Due in part to simultaneous detection of IgM and IgG for both S1 and N proteins, BioS exhibited the highest positive percent agreement at ≥11 days post-symptom onset (PSO). In conclusion, the BioS IgM/IgG rapid test was highly specific and demonstrated a higher positive percentage of agreement than all the enzyme-linked immunosorbent assay/chemiluminescence immunoassay (ELISA/CLIA) commercial tests considered in this study. Moreover, by detecting the presence of antibodies prior to 11 days PSO in 78.2% of the patients, the BioS test increased the efficiency of the diagnosis of SARS-CoV-2 infection in the early stages of the disease.

In their outstanding study, Wei et al 1 compared the intestinal microbiota (IM) of well-defined autoimmune hepatitis (AIH) patients to healthy controls. They reported significantly reduced IM alpha diversity and disparate IM compositional heterogeneity (beta diversity) with enrichment of Streptococcus , Veillonella , Klebsiella and Lactobacillus in AIH. The excellent achievement of this study is to have characterised the IM of untreated patients with AIH. However, differentiation between IM changes caused by AIH in particular and by chronic liver disease (CLD) in general was not possible because this study lacked a control group with CLD other than AIH. In a pilot study, we analysed the IM of patients with CLD of mixed aetiology in comparison to healthy controls to clarify alterations across different stages of CLD (table 1 and online supplementary data). In contrast to the study by Wei et al , patients with recent intake of antibiotics were excluded. Additionally, only patients on proton pump inhibitors (PPIs), which are frequently taken by patients with CLD, were included to avoid a bias due to mixed PPI use, because PPI can alter the IM, in particular, the abundance of Veillonellaceae and Streptococcaceae.2 Based on investigations of normalised relative reads (n-RR) per IM taxon as well as IM alpha-diversity and beta-diversity analyses, we detected Veillonella …

Bacteria from livestock may be opportunistic pathogens and carriers of clinically relevant resistance genes, as many antimicrobials are used in both veterinary and human medicine. They may be released into the environment from wastewater treatment plants (WWTPs), which are influenced by wastewater from slaughterhouses, thereby endangering public health. Moreover, process water that accumulates during the slaughtering of poultry is an important reservoir for livestock-associated multidrug-resistant bacteria and may serve as a vector of transmission to occupationally exposed slaughterhouse employees. Mitigation solutions aimed at the reduction of the bacterial discharge into the production water circuit as well as interventions against their further transmission and dissemination need to be elaborated. Furthermore, the efficacy of in-house WWTPs needs to be questioned. Reliable data on the occurrence and diversity of clinically relevant bacteria within the slaughtering production chain and in the WWTP effluents in Germany will help to assess their impact on public and environmental health., The wastewater of livestock slaughterhouses is considered a source of antimicrobial-resistant bacteria with clinical relevance and may thus be important for their dissemination into the environment. To get an overview of their occurrence and characteristics, we investigated process water (n = 50) from delivery and unclean areas as well as wastewater (n = 32) from the in-house wastewater treatment plants (WWTPs) of two German poultry slaughterhouses (slaughterhouses S1 and S2). The samples were screened for ESKAPE bacteria (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli. Their antimicrobial resistance phenotypes and the presence of extended-spectrum-β-lactamase (ESBL), carbapenemase, and mobilizable colistin resistance genes were determined. Selected ESKAPE bacteria were epidemiologically classified using different molecular typing techniques. At least one of the target species was detected in 87.5% (n = 28/32) of the wastewater samples and 86.0% (n = 43/50) of the process water samples. The vast majority of the recovered isolates (94.9%, n = 448/472) was represented by E. coli (39.4%), the A. calcoaceticus-A. baumannii (ACB) complex (32.4%), S. aureus (12.3%), and K. pneumoniae (10.8%), which were widely distributed in the delivery and unclean areas of the individual slaughterhouses, including their wastewater effluents. Enterobacter spp., Enterococcus spp., and P. aeruginosa were less abundant and made up 5.1% of the isolates. Phenotypic and genotypic analyses revealed that the recovered isolates exhibited diverse resistance phenotypes and β-lactamase genes. In conclusion, wastewater effluents from the investigated poultry slaughterhouses exhibited clinically relevant bacteria (E. coli, methicillin-resistant S. aureus, K. pneumoniae, and species of the ACB and Enterobacter cloacae complexes) that contribute to the dissemination of clinically relevant resistances (i.e., blaCTX-M or blaSHV and mcr-1) in the environment. IMPORTANCE Bacteria from livestock may be opportunistic pathogens and carriers of clinically relevant resistance genes, as many antimicrobials are used in both veterinary and human medicine. They may be released into the environment from wastewater treatment plants (WWTPs), which are influenced by wastewater from slaughterhouses, thereby endangering public health. Moreover, process water that accumulates during the slaughtering of poultry is an important reservoir for livestock-associated multidrug-resistant bacteria and may serve as a vector of transmission to occupationally exposed slaughterhouse employees. Mitigation solutions aimed at the reduction of the bacterial discharge into the production water circuit as well as interventions against their further transmission and dissemination need to be elaborated. Furthermore, the efficacy of in-house WWTPs needs to be questioned. Reliable data on the occurrence and diversity of clinically relevant bacteria within the slaughtering production chain and in the WWTP effluents in Germany will help to assess their impact on public and environmental health.

International audience; Acute respiratory tract infections (ARTI), including the common cold, pharyngitis, sinusitis, otitis media, bronchiolitis and pneumonia are the most common diagnoses among patients seeking medical care in western countries, and account for most antibiotic prescriptions. While a confirmed and fast ARTI diagnosis is key for antibiotic prescribing, empiric antimicrobial treatment remains common, because viral symptoms are often clinically similar and difficult to distinguish from those caused by bacteria. As a result, inappropriate antibiotic prescriptions are high and in certain settings likely higher than the commonly estimated 30%. The QIAstat Respiratory Panel® assay (QIAstat RP) is a multiplexed in vitro diagnostics test for the rapid simultaneous detection of 21 pathogens directly from respiratory samples, including human mastadenovirus A-G, primate bocaparvovirus 1+2, human coronavirus (HKU1, NL63, OC43, 229E), human metapneumovirus A/B, rhinovirus/enterovirus, influenza A virus (no subtype, subtype H1, H1N1/2009, H3), influenza B virus, human respirovirus 1+3, human orthorubulavirus 2+4, human orthopneumovirus, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. We describe the first multicenter study of 445 respiratory samples, collected through the 2016-2017 and 2018 respiratory seasons, with performance compared against BioFire FilmArray RP v1.7 and discrepancy testing by Seegene Allplex RP. The QIAstat RP demonstrated a positive percentage of agreement of 98.0% (95% CI: 96.0-99.1%) and a negative percentage agreement of 99.8% (95% CI: 99.6-99.9%). With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship may potentially be achieved.

Multidrug-resistant bacteria cause difficult-to-treat infections and pose a risk for modern medicine. Sources of multidrug-resistant bacteria include hospital, municipal and slaughterhouse wastewaters. In this study, bacteria with resistance to 3rd generation cephalosporins were isolated from all three wastewater biotopes, including a maximum care hospital, municipal wastewaters collected separately from a city and small rural towns and the wastewaters of two pig and two poultry slaughterhouses. The resistance profiles of all isolates against clinically relevant antibiotics (including β-lactams like carbapenems, the quinolone ciprofloxacin, colistin, and trimethoprim/sulfamethoxazole) were determined at the same laboratory. The bacteria were classified according to their risk to human health using clinical criteria, with an emphasis on producers of carbapenemases, since carbapenems are prescribed for hospitalized patients with infections with multi-drug resistant bacteria. The results showed that bacteria that pose the highest risk, i. e., bacteria resistant to all β-lactams including carbapenems and ciprofloxacin, were mainly disseminated by hospitals and were present only in low amounts in municipal wastewater. The isolates from hospital wastewater also showed the highest rates of resistance against antibiotics used for treatment of carbapenemase producers and some isolates were susceptible to only one antibiotic substance. In accordance with these results, qPCR of resistance genes showed that 90% of the daily load of carbapenemase genes entering the municipal wastewater treatment plant was supplied by the clinically influenced wastewater, which constituted approximately 6% of the wastewater at this sampling point. Likewise, the signature of the clinical wastewater was still visible in the resistance profiles of the bacteria isolated at the entry into the wastewater treatment plant. Carbapenemase producers were not detected in slaughterhouse wastewater, but strains harboring the colistin resistance gene mcr-1 could be isolated. Resistances against orally available antibiotics like ciprofloxacin and trimethoprim/sulfamethoxazole were widespread in strains from all three wastewaters.

The high use of antibiotics in human and veterinary medicine has led to a wide spread of antibiotics and antimicrobial resistance into the environment. In recent years, various studies have shown that antibiotic residues, resistant bacteria and resistance genes, occur in aquatic environments and that clinical wastewater seems to be a hot spot for the environmental spread of antibiotic resistance. Here a representative statistical analysis of various sampling points is presented, containing different proportions of clinically influenced wastewater. The statistical analysis contains the calculation of the odds ratios for any combination of antibiotics with resistant bacteria or resistance genes, respectively. The results were screened for an increased probability of detecting resistant bacteria, or resistance genes, with the simultaneous presence of antibiotic residues. Positive associated sets were then compared, with regards to the detected median concentration, at the investigated sampling points. All results show that the sampling points with the highest proportion of clinical wastewater always form a distinct cluster concerning resistance. The results shown in this study lead to the assumption that ciprofloxacin is a good indicator of the presence of multidrug resistant P. aeruginosa and extended spectrum β-lactamase (ESBL)-producing Klebsiella spec., Enterobacter spec. and Citrobacter spec., as it positively relates with both parameters. Furthermore, a precise relationship between carbapenemase genes and meropenem, regarding the respective sampling sites, could be obtained. These results highlight the role of clinical wastewater for the dissemination and development of multidrug resistance.

Bordetella trematum is a relatively newly discovered and potentially frequently overlooked Bordetella species, mostly isolated from chronic wounds and predominantly in those of the lower extremities. Its susceptibility profile and clinical significance is still debated, given the limited amount of available data. We contribute providing a molecular and phenotypical analysis of three unique clinical B. trematum isolates detected between August 2019 and January 2020 to aid the matter. Cryo-conserved isolates were subcultured and re-identified using various routine means of identification. Bacterial genomes were fully Illumina-sequenced and phenotypical susceptibility was determined by broth microdilution and gradient-strip tests. All isolates displayed increased susceptibility to piperacillin–tazobactam (B. trematum can be resistant to trimethoprim/sulfamethoxazole is warranted.

Background Experimental animal models are indispensable components of preclinical sepsis research. Reproducible results highly rely on defined and invariant baseline conditions. Our hypothesis was that the murine gut microbiota varies among different distributors of laboratory animals and that these variations influence the phenotype of abdominal sepsis derived from a bacterial inoculum model (intraperitoneal stool injection). Materials and methods Male C57BL/6 mice (8-wk old) purchased from Charles River (CR), Janvier (J), and Harlan (H) were sacrificed, and the bacterial composition of feces was analyzed using CHROMagar orientation medium. Stool was injected intraperitoneally into CR mice, followed by clinical observation and gene expression analysis. Experiments were repeated 16 mo later under the same conditions. Results Stool analysis revealed profound intervendor differences in bacterial composition, mainly regarding Staphylococcus aureus and Bacillus licheniformis . Mice challenged with CR as well as H feces developed significantly higher severity of disease and died within the observation period, whereas stool from J mice did not induce any of these symptoms. Real-time polymerase chain reaction revealed corresponding results with significant upregulation of proinflammatory cytokines and vascular leakage-related mediators in CR and H injected animals. Sixteen months later, the bacterial fecal composition had significantly shifted. The differences in clinical phenotype of sepsis after intraperitoneal stool injection had vanished. Conclusions We are the first to demonstrate vendor and time effects on the murine fecal microbiota influencing sepsis models of intraabdominal stool contamination. The intestinal microbiota must be defined and standardized when designing and interpreting past and future studies using murine abdominal sepsis models.

Acinetobacter baumannii is an important human pathogen usually associated with severe hospital-acquired infections. Here, we announce the draft genome sequences of two livestock-associated isolates recovered from sewage water from a poultry slaughterhouse in Germany. Short-read whole-genome sequencing was conducted to determine the genetic basis of their antimicrobial resistance phenotype.

In acute gastroenteritis (AGE), identification of the infectious agent is important for patient management. Since symptoms do not reliably identify the agent, microbiological diagnostics are important. Conventional methods lack sensitivity and often take days. Multiplex PCR panels offer fast and sensitive alternatives. Our aim was to assess the performance of the new QIAstat Gastrointestinal Panel (GIP) detecting 24 different gastroenteric pathogens from stool in Cary-Blair transport medium (Adenovirus F 40/41, Astrovirus, Norovirus GI/GII, Rotavirus A, Sapovirus, Campylobacter spp., Clostridium difficile, Plesiomonas shigelloides, Salmonella spp., Vibrio cholera, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, enteroaggregative Escherichia coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica and Giardia lamblia). We tested both prospective (n = 163) and retrospective (n = 222) stool samples sent for routine diagnostics by the QIAstat GIP comparing it to the FDA-approved BioFire FilmArray GIP. Seegene Allplex GIP was used for discrepancy testing. After discrepancy testing, QIAstat GIP detected 447 of 455 pathogens (98.2%, 95% confidence interval (CI) 96.6–99.1%). There were eight false positive detections. Multiple pathogens were detected in 32.5% of positive samples. The QIAstat GIP detected a large range of AGE pathogens with a high sensitivity. It offers an easy-to-use system for GI pathogen detection in stool within 70 min. An advantage of the QIAstat is the availability of cycle threshold (CT) values to aid in interpretation of results.

Increasing isolation rates of resistant bacteria in the last years require identification of potential infection reservoirs in healthcare facilities. Especially the clinical wastewater network represents a potential source of antibiotic resistant bacteria. In this work, the siphons of the sanitary installations from 18 hospital rooms of two German hospitals were examined for antibiotic resistant bacteria and antibiotic residues including siphons of showers and washbasins and toilets in sanitary units of psychosomatic, haemato-oncological, and rehabilitation wards. In addition, in seven rooms of the haemato-oncological ward, the effect of 24 h of stagnation on the antibiotic concentrations and MDR (multi-drug-resistant) bacteria in biofilms was evaluated. Whereas no antibiotic residues were found in the psychosomatic ward, potential selective concentrations of piperacillin, meropenem and ciprofloxacin were detected at a rehabilitation ward and ciprofloxacin and trimethoprim were present at a haemato-oncology ward. Antibiotic resistant bacteria were isolated from the siphons of all wards, however in the psychosomatic ward, only one MDR strain with resistance to piperacillin, third generation cephalosporins and quinolones (3MRGN) was detected. In contrast, the other two wards yielded 11 carbapenemase producing MDR isolates and 15 3MRGN strains. The isolates from the haemato-oncological ward belonged mostly to two specific rare sequence types (ST) (P. aeruginosa ST823 and Enterobacter cloacae complex ST167). In conclusion, clinical wastewater systems represent a reservoir for multi-drug-resistant bacteria. Consequently, preventive and intervention measures should not start at the wastewater treatment in the treatment plant, but already in the immediate surroundings of the patient, in order to minimize the infection potential.

Slaughterhouse process- and wastewater are considered as a hotspot for antibiotic-resistant bacteria and antimicrobial residues and may thus play an important role for their dissemination into the environment. In this study, we investigated occurrence and characteristics of ESKAPE bacteria (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp.) and ESBL (extended spectrum β-lactamase) -producing E. coli in water samples of different processing stages of two German pig slaughterhouses (S1/S2) as well as their municipal wastewater treatment plants (mWWTPs). Furthermore, residues of various antimicrobials were determined. A total of 103 water samples were taken in delivery and dirty areas of the slaughterhouses S1/S2 (n = 37), their in-house WWTPs (n = 30) and mWWTPs including their receiving water bodies (n = 36). The recovered isolates (n = 886) were characterized for their antimicrobial resistance pattern and its genetic basis. Targeted species were ubiquitous along the slaughtering and wastewater chains. Phenotypic and genotypic analyses revealed a broad variety of resistance phenotypes and β-lactamase genes. Carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE) and healthcare-associated (HA) MRSA were recovered only from mWWTPs and their preflooders. In contrast, the mcr-1 gene was exclusively detected in E. coli from S1/S2. Residues of five antimicrobials were detected in 14.9% (10/67) of S1/S2 samples in low range concentrations (≤1.30 μg/L), whereas 91.7% (33/36) of mWWTPs samples exhibited residues of 22 different antibiotics in concentrations of up to 4.20 μg/L. Target bacteria from S1/S2 and mWWTPs exhibited differences in their abundances, resistance phenotypes and genotypes as well as clonal lineages. S1/S2 samples exhibited bacteria with zoonotic potential (e.g. MRSA of CC398, E. coli of significant clones), whereas ESKAPE bacteria exhibiting resistances of clinical importance were mainly detected in mWWTPs. Municipal WWTPs seem to fail to eliminate these bacteria leading to a discharge into the preflooder and a subsequent dissemination into the surface water.

Carbapenem antibiotics constitute the mainstay therapy of nosocomial infections with extended spectrum beta-lactamase producing Gram-negative bacteria; however, resistance against these compounds is increasing. This study was designed to demonstrate that carbapenemase-producing bacteria are disseminated from hospitals into the environment. To this end, resistant bacteria were isolated from a clinical/urban and from a rural catchment system in Germany in 2016/17. The study followed the dissemination of resistant bacteria from the wastewater through the wastewater treatment plant (WWTP) into the receiving surface waters. The bacteria were cultivated on selective agar and characterized by antibiotic testing, real-time PCR targeting carbapenemase genes and typing. Bacteria with resistance to third generation cephalosporins were isolated from all sample sites. 134 isolates harboring carbapenemase genes encoding VIM, NDM and OXA-48 and 26 XDR (extensively drug-resistant) strains with susceptibility to only one or two antibiotics were isolated from the clinical/urban system. The rural system yielded eight carbapenemase producers and no XDR strains. In conclusion, clinical wastewaters were charged with a high proportion of multidrug resistant bacteria. Although most of these bacteria were eliminated during wastewater treatment, dissemination into surface waters is possible as single carbapenemase producers were still present in the effluent of the WWTP.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411-2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and counter-regulator of endogenous glucocorticoids (GCs). It is implicated in acute and chronic inflammatory diseases. This study investigated the role of the MIF-GC regulatory dyad in the expression and release of matrix metalloproteinase-2 (MMP-2) during periodontitis, in vivo and in vitro. In a Mif-knockout (KO) mouse model of ligature-induced periodontitis, gingival tissues and blood were collected and analysed for levels of interleukin-6 (IL-6), MIF, MMP-2, and corticosterone. In addition, human gingival fibroblasts (HGFs) were tested for production of IL-6 and MMP-2 after stimulation with hydrocortisone (HC), MIF, tumour necrosis factor-alpha (TNF-α), or Fusobacterium nucleatum, a pathogen known to elicit immune responses during periodontitis. Wild-type (WT) mice showed a local and systemic increase of MIF levels during inflammation, which was confirmed by increased local IL-6 concentrations. Systemic GC levels were reduced in WT and Mif-KO mice during inflammation, with overall lower concentrations in Mif-KO mice. In vivo and in vitro, MMP-2 production was not dependent on MIF or inflammatory stimuli, but was inhibited by HC. Therefore, MIF does not appear to stimulate expression of MMP-2 in the gingival tissues, whereas GC upregulates MIF and downregulates MMP-2. Our findings further suggest that MIF may regulate systemic GC levels.

Detection of intestinal protozoan parasites by light microscopy is cumbersome, needs experienced personnel, and may lack sensitivity and/or specificity as compared with molecular-based stool assays. Here, we evaluated the BD MAX™ Enteric Parasite Panel, i.e., a multiplex real-time PCR assay for simultaneous detection of Giardia duodenalis, Entamoeba histolytica, and cryptosporidia (Cryptosporidium parvum and C. hominis), by examining 200 positive human stool samples (138 × G. duodenalis, 27 × E. histolytica, 35 × Cryptosporidium spp.) and 119 controls including 18 samples with E. dispar. The majority of the samples, i.e., 153/200 (76.5%) positive samples and 66/119 (55.5%) controls, were confirmed by multiplex in-house PCR detecting the same parasites as the BD MAX™ Enteric Parasite Panel. The BD MAX™ assay did not yield false-positive results. Sensitivity and specificity were 97.8% (95% CI, 93.3-99.4%) and 100% (95% CI, 97.4-100%) for G. duodenalis, 100% (95% CI, 84.5-100%) and 100% (95% CI, 98.4-100%) for E. histolytica, and 100% (95% CI, 87.7-100%) and 100% (95% CI, 98.3-100%) for cryptosporidia, and similar data were obtained when only the 219 PCR-confirmed samples were analyzed. Thus, the BD MAX™ Enteric Parasite Panel provides a highly sensitive and specific tool for the laboratory diagnosis of three predominant protozoan parasites causing enteritis.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) recognizes muramyl dipeptide (MDP) of bacterial cell walls, triggering NFκB-induced pro-inflammation. As most human pathogenic filariae contain Wolbachia endobacteria that synthesize the MDP-containing cell wall precursor lipid II, NOD2’s role during infection with the rodent filaria Litomosoides sigmodontis was investigated. In NFκB reporter-cells, worm-extract containing Wolbachia induced NOD2 and NOD1. NOD2-deficient mice infected with L. sigmodontis had significantly more worms than wildtype controls early in infection. Increased worm burden was not observed after subcutaneous infection, suggesting that protective NOD2-dependent immune responses occur within the skin. Flow cytometry demonstrated that neutrophil recruitment to the skin was impaired in NOD2−/− mice after intradermal injection of third stage larvae (L3), and blood neutrophil numbers were reduced after L. sigmodontis infection. PCR array supported the requirement of NOD2 for recruitment of neutrophils to the skin, as genes associated with neutrophil recruitment and activation were downregulated in NOD2−/− mice after intradermal L3 injection. Neutrophil depletion before L. sigmodontis infection increased worm recovery in wildtype mice, confirming that neutrophils are essential against invading L3 larvae. This study indicates that NOD-like receptors are implemented in first-line protective immune responses against filarial nematodes.

Background Spontaneous bacterial peritonitis (SBP) can be life-threatening in patients with liver cirrhosis. In contrast to community-acquired SBP, no standard treatment has been established for healthcare-related and nosocomial SBP. Patients and methods We prospectively collected healthcare-related and nosocomial SBP cases from March 2012 till February 2016 at the Department of Internal Medicine I of the University of Bonn and analysed the prevalence of antibiotic resistance among the isolated bacteria. SBP was diagnosed according to international guidelines. Ciprofloxacin, ceftriaxone and meropenem were used as reference substance for resistance to quinolones, third-generation cephalosporins and carbapenems, respectively. Results Ninety-two SBP episodes in 86 patients were identified: 63 episodes (69%) were nosocomial. Escherichia coli, Klebsiella species, enterococci and streptococci were most frequently isolated. Frequency of these microorganisms were comparable for healthcare-related and nosocomial SBP (14 vs. 11%, 14 vs. 8%, 14 vs. 5% and 10 vs. 6%, respectively). In general, antibiotic resistance was higher in isolates from nosocomial than from healthcare-related SBP (50% vs. 18% for quinolones, 30 vs. 11% for piperacillin-tazobactam; p>0.05), but comparable concerning third-generation cephalosporins (30 vs. 33%). All microorganisms were sensitive to carbapenems apart from nosocomial infections with Enterococcus faecium (n=3) and Candida albicans (n=1) due to intrinsic resistance or lack of microbiological efficacy, respectively. No multidrug-resistant microorganisms were detected. Resistance to initial antibiotic treatment affected 30-day survival negatively (18% vs. 68%; p=0.002). Conclusion Resistance to initial antibiotic treatment was associated with increased mortality. With resistance to cephalosporins being frequent, piperacillin-tazobactam or carbapenems might be preferred as treatment of SBP. This article is protected by copyright. All rights reserved.

Background : Aggressive periodontitis is associated with the presence of Aggregatibacter actinomycetemcomitans , a leukotoxin (Ltx)-producing periodontal pathogen. Ltx has the ability to lyse white blood cells including neutrophils. Objectives : This study was aimed at investigating the interactions between neutrophils and Ltx with regard to the chemotactic properties of Ltx and the release of neutrophil extracellular traps (NETs). Methods : Neutrophils from healthy blood donors were isolated and incubated for 30 min and 3 h with increasing concentrations of Ltx (1, 10, and 100 ng/mL) as well as with A. actinomycetemcomitans strains (NCTC 9710 and HK 1651) producing different levels of Ltx. Formation of NETs and cell lysis were assessed by microscopy, fluorescence-based assays, and measurement of released lactate dehydrogenase. Neutrophil migration in response to different Ltx gradients was monitored by real-time video microscopy, and image analysis was performed using ImageJ software. Results : Although Ltx (10 and 100 ng/mL) and the leukotoxic A. actinomycetemcomitans strain HK 1651 lysed some neutrophils, other cells were still capable of performing NETosis in a concentration-dependent manner. Low doses of Ltx and the weakly leukotoxic strain NCTC 9710 did not lead to neutrophil lysis, but did induce some NETosis. Furthermore, all three concentrations of Ltx enhanced random neutrophil movement; however, low directional accuracy was observed compared with the positive control (fMLP). Conclusions : The results indicate that Ltx acts both as a neutrophil activator and also causes cell death. In addition, Ltx directly induces NETosis in neutrophils prior to cell lysis. In future studies, the underlying pathways involved in Ltx-meditated neutrophil activation and NETosis need to be investigated further. Keywords: leukotoxin; Aggregatibacter actinomycetemcomitans; neutrophils; NETs; migration; chemotaxis; aggressive periodontitis (Published: 8 November 2016) Citation: Journal of Oral Microbiology 2016, 8: 33070 - http://dx.doi.org/10.3402/jom.v8.33070

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells. Background: Human B cells respond to Toll-like receptor (TLR) 9 stimulation by cytokine production. Results: A rare, novel TLR9 allele fails to activate NF-κB in HEK293 cells. Heterozygous carrier B cells show defective IL-6 and IL-10 production. Conclusion: Heterozygosity for this TLR9 allele modifies CpG oligonucleotide responsiveness. Significance: This is the first analysis of human TLR9 variants in primary cells.

In previous studies we reported that plasmacytoid dendritic cells (PDC) infiltrating head and neck cancer tissue are functionally impaired, but the molecular basis for the functional deficiency remained unclear. Here we demonstrate that tumour-derived prostaglandin E2 (PGE(2)) and transforming growth factor-beta (TGF-beta) increase interleukin-8 (IL-8) but synergistically inhibit interferon-alpha (IFN-alpha) and tumour necrosis factor (TNF) production of Toll-like receptor 7 (TLR7)- and Toll-like receptor 9 (TLR9)-stimulated PDC. The inhibitory effect of PGE(2) could be mimicked by the induction of cyclic AMP (cAMP) and by inhibitors of cyclooxygenase. The contribution of tumour-derived TGF-beta was confirmed by the TGF-beta antagonist SB-431542. Suppression of tumour-derived PGE(2) and TGF-beta restored TLR-induced IFN-alpha production of PDC. Additionally, PGE(2)- and TGF-beta-treated PDC display a 'tolerogenic' phenotype because of a downregulation of CD40 accompanied by an upregulation of CD86. Finally, in TLR-stimulated PDC, PGE(2) and TGF-beta reduce the CCR7:CXCR4 ratio, suggesting that PDC are impaired in their ability to migrate to tumour-draining lymph nodes but are retained in stromal cell-derived factor 1 (SDF-1)-expressing tissues. Based on these data, cyclooxygenase inhibitors and TGF-beta antagonists may improve TLR7- and TLR9-based tumour immunotherapy.

Type I IFNs represent a major antimicrobial defense mechanism due to their property of enhancing immune responses by priming both innate and adaptive immune cells. Plasmacytoid dendritic cells (pDC) are the major source of type I IFN in the human body and represent innate immune cells involved in first-line defense against invading pathogens. Although pDC activation has been extensively studied upon stimulation with synthetic TLR ligands, viruses, and intracellular bacteria, there is only scarce information on extracellular bacteria. In this study we show that the triggering of human pDC-derived IFN-α secretion by Staphylococcus aureus is independent of TLR2 and specific for coagulase-positive staphylococci. Specificity of the pDC response to S. aureus is independent of the bacterial virulence factors protein A and α-toxin but is mediated by Ag-specific IgG and CD32. S. aureus-induced pDC activation can be blocked by inhibitory DNA oligonucleotides and chloroquine, suggesting that engagement of TLR7/9 by bacterial nucleic acids after CD32-mediated uptake of these compounds may play a central role in this process. Altogether, we propose that in marked contrast to nonselective TLR2-dependent activation of most innate immune cells, pDC activation by S. aureus represents an Ag-specific memory response since it requires the presence of class-switched immunoglobulins.