Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below USD700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed ‘lab-on-a-comb’ to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 ‘lab-on-a-comb’ devices to be imaged which is equivalent 3480 individual 1μl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes

Bacterial Community Profiling of Milk Samples as a Means to Understand Culture-Negative Bovine Clinical Mastitis

Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease

Strategische Ansätze zur Bekämpfung von Mykoplasmenmastitiden

Bovine mastitis caused by Mycoplasma and in particular by Mycoplasma (M.) bovis is a serious animal-health problem for (bigger) dairy herds. It is a highly contagious disease with low infectious doses and with high amounts of shedding This can lead to a rapid dissemination of the infections in a herd followed by milk yield decline. After appearance of typical clinical cases the disease is often self-limiting, disappearing within months of outbreaks sometimes without intervention. Intramammary infections with Mycoplasma are largely untreatable by chemotherapy. All recommended control measures are based on the prevention of new infections of the mammary glands by identification and removal of infected animals from the herd. Differences exist in the selection of animals to be examined (all animals of the herd or subgroups) and in the type of removal of the animals from the herd (slaughter or separation). Because a risk assessment of different strategic measures is not possible yet, different strategic approaches are used in the world. The article explains the different approaches

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8 mu g/mL for erythromycin, >16 mu g/mL for spiramycin, and >32 mu g/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32 mu g/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC = 0.5 mu g/mL and 30 isolates >32 mu g/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5 mu g/mL) and 1-fold higher MIC90 (4 vs. 2 mu g/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2 mu g/mL, whereas tiamulin had a MIC90 of 0.5 mu g/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population