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.

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.

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.

SummaryIn 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-b (TGF-b) increase interleukin-8 (IL-8) but synergistically inhibit interferon-a (IFN-a) 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-b was confirmed by the TGF-b antagonist SB-431542. Suppression of tumour-derived PGE 2 and TGF-b restored TLR-induced IFN-a production of PDC. Additionally, PGE 2 -and TGF-b-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-b 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-b antagonists may improve TLR7-and TLR9-based tumour immunotherapy.

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Livestock-associated bacteria with resistance to two or more antibiotic drug classes have heightened our awareness for the consequences of antibiotic consumption and spread of resistant bacterial strains in the veterinary field. In this study we assessed the prevalence of concomitant colonization with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and enterobacteriaceae expressing extended-spectrum betalactamases (ESBL-E) in farms at the German-Dutch border region. Nasal colonization of pigs with MRSA (113/547 (20.7%)) was less frequent than rectal colonization with ESBL-E (163/540 (30.2%)). On the individual farm level MRSA correlated with ESBL-E recovery. The data further provide information on prevalence at different stages of pig production, including abattoirs, as well as in air samples and humans living and working on the farms. Notably, MRSA was detected in stable air samples of 34 out of 35 pig farms, highlighting air as an important MRSA transmission reservoir. The majority of MRSA isolates, including those from humans, displayed tetracycline resistance and spa types t011 and t034 characteristic for LA-MRSA, demonstrating transmission from pigs to humans. ESBL-E positive air samples were detected on 6 out of 35 farms but no pig-to-human transmission was found. Detection of ESBL-E, e.g. mostly Escherichia coli with CTX-M-type ESBL, was limited to these six farms. Molecular typing revealed transmission of ESBL-E within the pig compartments; however, related strains were also found on unrelated farms. Although our data suggest that acquisition of MRSA and ESBL-E might occur among pigs in the abattoirs, MRSA and ESBL-E were not detected on the carcasses. Altogether, our data define stable air (MRSA), pig compartments (ESBL-E) and abattoir waiting areas (MRSA and ESBL-E) as major hot spots for transmission of MRSA and/or ESBL-E along the pig production chain.

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.

BackgroundAggressive 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.ObjectivesThis 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).MethodsNeutrophils 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.ResultsAlthough 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).ConclusionsThe 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.