Direct-PCR from rectal swabs and environmental reservoirs: A fast and efficient alternative to detect blaOXA-48 carbapenemase genes in an Enterobacter cloacae outbreak setting.

Carbapenemase-producing bacteria are a risk factor in clinical settings worldwide. The aim of the study was to accelerate the time to results during an outbreak situation with blaOXA-48-positive Enterobacter cloacae by using a real-time multiplex quantitative PCR (qPCR) directly on rectal swab specimens and on wastewater samples to detect carbapenemase-producing bacteria. Thus, we analyzed 681 rectal swabs and 947 environmental samples during a five-month period by qPCR and compared the results to culture screening. The qPCR showed a sensitivity of 100% by testing directly from rectal swabs and was in ten cases more sensitive than the culture-based methods. Environmental screening for blaOXA-48-carbapenemase genes by qPCR revealed reservoirs of different carbapenemase genes that are potential sources of transmission and might lead to new outbreaks. The rapid identification of patients colonized with those isolates and screening of the hospital environment is essential for earlier patient treatment and eliminating potential sources of nosocomial infections.

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

BACKGROUND: Undetected 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. AIM: The 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. METHODS: We 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). FINDINGS: Four 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. CONCLUSION: Implementing 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.

Effects of metal exposure on motor neuron development, neuromasts and the escape response of zebrafish embryos.

Low level metal contaminations are a prevalent issue with often unknown consequences for health and the environment. Effect-based, multifactorial test systems with zebrafish embryos to assess in particular developmental toxicity are beneficial but rarely used in this context. We therefore exposed wild-type embryos to the metals copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) for 72 h to determine lethal as well as sublethal morphological effects. Motor neuron damage was investigated by immunofluorescence staining of primary motor neurons (PMNs) and secondary motor neurons (SMNs). In vivo stainings using the vital dye DASPEI were used to quantify neuromast development and damage. The consequences of metal toxicity were also assessed functionally, by testing fish behavior following tactile stimulation. The median effective concentration (EC50) values for morphological effects 72 h post fertilization (hpf) were 14.6 mg/L for cadmium and 0.018 mg/L for copper, whereas embryos exposed up to 45.8 mg/L cobalt showed no morphological effects. All three metals caused a concentration-dependent reduction in the numbers of normal PMNs and SMNs, and in the fluorescence intensity of neuromasts. The results for motor neuron damage and behavior were coincident for all three metals. Even the lowest metal concentrations (cadmium 2mg/L, copper 0.01 mg/L and cobalt 0.8 mg/L) resulted in neuromast damage. The results demonstrate that the neuromast cells were more sensitive to metal exposure than morphological traits or the response to tactile stimulation and motor neuron damage.