Frank Günther

Host defence against Staphylococcus aureus biofilms infection: Phagocytosis of biofilms by polymorphonuclear neutrophils (PMN)

Bacteria organised in biofilms are a common cause of relapsing or persistent infections, particularly in patients receiving medical implants such as ventilation tubes, indwelling catheters, artificial heart valves, endoprostheses, or osteosynthesis materials. Bacteria in biofilms are relatively resistant towards antibiotics and biocides, and--according to the current dogma--towards the host defence mechanisms as well. In that context, we addressed the question, how polymorphonuclear neutrophils (PMN), the first line defence against bacterial infection, would interact with Staphylococcus aureus biofilms generated in vitro. By time-lapse video microscopy and confocal laser scan microscopy we observed a migration of PMN towards and into the biofilms, as well as clearance of biofilms by phagocytosis. By labelling the bacteria within the biofilm with (3)H thymidine, and by cytofluorometry we could confirm and quantify clearance and phagocytosis of biofilm as well. Of note, the extent of biofilm clearance depended on its maturation state: developing young biofilms were more sensitive towards the attack by PMN compared to mature biofilms. In conclusion, contrary to the current dogma, S. aureus biofilms are not inherently protected against the host defence.

Expression of the CXCR6 on polymorphonuclear neutrophils in pancreatic carcinoma and in acute, localized bacterial infections

The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T‐cells to non‐lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up‐regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFα) was seen: brief exposure with low‐dose TNFα induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up‐regulated under inflammatory conditions and mediates migration of CXCR6‐positive PMN.

Host defence against Staphylococcus aureus biofilms by polymorphonuclear neutrophils: oxygen radical production but not phagocytosis depends on opsonisation with immunoglobulin G.

Bacterial biofilms are increasingly recognised as a major cause of persistent infection and destructive inflammatory processes. In patients with biofilm infection, massive infiltration of leukocytes, particularly polymorphonuclear neutrophils is seen, and previous in vitro studies showed that PMN were able to phagocytose Staphylococcus aureus biofilms. We now addressed the question whether opsonisation of biofilms with immunoglobulin G and complement enhances the efficiency of phagocytosis, as it has been shown for free-living planktonic bacteria and other particulate materials. We found that incubation of biofilms with normal human serum resulted in IgG binding and in complement activation with deposits on the biofilm of C3bi. This opsonisation, however, did not affect the adherence of PMN to the biofilms nor did it enhance degranulation or phagocytosis. The clearance of biofilms, however, was increased, and the oxygen radical production by the PMN depended critically on the coating of biofilms with IgG.

Expression of Galectin-3 in Pancreatic Ductal Adenocarcinoma

Galectin-3 influences neoangiogenesis, tumor cell adhesion, and tumor-immune-escape mechanisms. Hence, the expression of galectin-3 in pancreatic ductal adenocarcinoma (PDAC) was evaluated. Galectin-3 expression in PDAC cell lines was proven by the presence of intracellular protein and by release into the supernatant. Furthermore, galectin-3 was found in the majority of human tissue samples. Serum concentrations of galectin-3 in PDAC patients did not differ significantly from healthy donors and did not correlate with established tumor markers. In conclusion, galectin-3 is expressed in PDAC tissues suggesting a role in tumor development; however, no relationship between expression and clinical findings could be established.

Natural isothiocyanates express antimicrobial activity against developing and mature biofilms of Pseudomonas aeruginosa

BACKGROUNDnThe antimicrobial properties of natural isothiocyanates (ITCs) found in plants such as nasturtium (Tropaeolum majus) and horseradish (Armoracia rusticana), and the need of new chemotherapeutic options for treatment of infections caused by multidrug-resistant and biofilm-forming Gram-negative bacteria such as Pseudomonas aeruginosa (Pa), led us to evaluate the effects of three major ITCs, allylisothiocyanate (AITC), benzylisothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC), and a mixture (ITCM) adapted to the ITC composition after release of active components out of natural sources.nnnMATERIAL/METHODSnOut of 105Pa isolates 27 isolates with increased biofilm formation were selected for testing. The effects of ITCs on Pa were evaluated regarding (1) planktonic bacterial proliferation, (2) biofilm formation, (3) metabolic activity in mature biofilms, and (4) synergism of ITCs and antibiotics.nnnRESULTSn(1) Each ITC had anti-Pa activity. Mean minimum inhibitory concentrations (MICs) were (μg/ml, mean±standard deviation): AITC 103±6.9; BITC, 2145±249; PEITC 29,423±1652; and ITCM, 140±5. (2) Treating bacteria with PEITC and ITCM in concentrations below the MIC significantly inhibited biofilm formation. Particularly, ITCM reduced biofilm mass and bacterial proliferation. (3) ITCs significantly inhibited metabolic activity in mature biofilms. (4) Combining ITCs with meropenem synergistically increased antimicrobial efficacy on Pa biofilms.nnnCONCLUSIONSnITCs represent a promising group of natural anti-infective compounds with activity against Pa biofilms.

Device-related infections in long-term healthcare facilities: the challenge of prevention

The world is aging and the number of elderly multimorbid patients is steadily increasing. The limited numbers of acute care beds in hospitals, in addition to the need to reduce costs, has led to the introduction of efficient discharge policies, which in turn have increased demand for beds in nursing homes and long-term care facilities (LTCFs). As a consequence, the number of postacute LTCF residents is rising, as is the number of residents requiring complex medical care delivered by use of indwelling medical devices. These devices place patients at a heightened risk for infection. Furthermore, infection control resources in LTCFs are often limited. This article reviews the preventive measures that should be taken in LTCFs to reduce the risk of device-related infections.

Comparative testing of disinfectant efficacy on planktonic bacteria and bacterial biofilms using a new assay based on kinetic analysis of metabolic activity.

The aim of our study was to develop a new reproducible method for disinfectant efficacy testing on bacterial biofilms and to evaluate the efficacy of different disinfectants against biofilms. Clinical multidrug‐resistant strains were chosen as test isolates to ensure practical relevance.

Sterility Testing of Injectable Products: Evaluation of the Growth-based BacT/ALERT® 3D™ Dual T Culture System

Sterility testing as described in the European Pharmacopoeia Chapter 2.6.1 as well as the United States Pharmacopeia Chapter 71 requires a 14 day incubation period of the test product in two different media and at two different temperatures. Because of extensive personnel requirements for test performance and quality assurance, alternative and partially automated methods for product sterility testing are of interest. The study objective was to evaluate the applicability of the BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microbial contaminants according to current pharmacopoeia standards. In addition, we compared the BacT/ALERT® 3D™ Dual T system to conventional pharmacopoeia sterility testing using the direct inoculation method. The results showed no significant disadvantages of sterility testing by BacT/ALERT® 3D™ Dual T compared to the direct inoculation method regarding the ability to detect microbial contamination. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products of sample volume below 10 mL and without antimicrobial activity. LAY ABSTRACT: Sterility is defined as the freedom from the presence of viable microorganisms. Injectable pharmaceutical products that are sometimes used for treatment of patients need to be free of microorganisms and therefore tested in the laboratory to confirm sterility. The testing procedures are described in detail in the European Pharmacopoeia. To test for sterility, the products need to be incubated for 14 days at two different temperatures and using two different growth media to ensure growth of various microorganisms. These tests, however, are cost- and labor-intensive. Therefore, we sought to use an automated system to reduce costs and hands-on time, as well as to improve quality assurance. The study objective was to evaluate the applicability of the automated BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microorganisms according to current pharmacopeia standards. We compared the BacT/ALERT® 3D™ Dual T system to standard sterility testing using the so-called direct inoculation method. The results showed no major disadvantages of sterility testing by the automated system compared to the standard method with regard to the detection level of microorganisms. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Additionally, the new automated method provided reliable results and promises a higher robustness to human errors than do standard manual methods, which might reduce the potential for errors and improves quality assurance. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products.

Improvement of Hand Hygiene Quality and Compliance Using Bioburden Measurement and Online Feedback in Germany.

To improve compliance with hand hygiene, a novel method with inclusion of an online reporting system was developed, comprising measurement of total hand bioburden, anonymous online feedback, and onsite training. The intervention significantly improved both compliance and quality of hand hygiene and reduced Staphylococcus aureus incidence. Infect Control Hosp Epidemiol 2016;1-4.

Improvement of infection control management by routine molecular evaluation of pathogen clusters

BACKGROUNDnUndetected pathogen clusters can often be a source of spreading in-hospital infections. Unfortunately, detection of clusters can be problematic because epidemiological connection is not always easily established. Infection prevention and control (IPC) measures, however, are most effective when applied at the earliest possible stage.nnnAIMnThe goal of our study was to evaluate the benefits of routine use of molecular typing techniques for IPC management in a large University teaching hospital.nnnMETHODSnWe implemented daily routine molecular typing of pathogen clusters using cost-effective standard methods such as random amplified polymorphic DNA PCR, multiple-locus variable number tandem repeat analysis, and spa-typing over a 4-year study period (2012-2015).nnnFINDINGSnFour pathogen clusters were evaluated: (I) 14 cases of Clostridium difficile in a peripheral ward, (II) 17 cases of methicillin-resistant Staphylococcus aureus (MRSA) in two intensive-care units (ICUs), (III) 21 cases of multidrug-resistant Klebsiella pneumoniae within one department, and (IV) 6 cases of vancomycin-resistant Enterococcus faecium in an interdisciplinary ICU. Typing revealed that cluster I was not caused by an outbreak strain but was likely due to different endogenous infections. Clusters III and IV showed a classical space-time clustering of point source outbreaks. Cluster II represented a prolonged temporal cluster, which would have gone undetected without molecular typing because of large intercase intervals.nnnCONCLUSIONnImplementing daily routine molecular typing is effective for detecting and analyzing pathogen clusters. Falsely suspected outbreaks can be quickly resolved, whereas actual outbreaks can be identified faster, so that targeted IPC measures can be applied earlier.