Title: Metabolic Profiling of Borrelia in Ticks and in Whole Blood Samples by Headspace Gas Chromatography - A Case Report

Abstract:

The technique of static headspace gas chromatography (HS-GC) was applied to monitor the emission of hydrogen and carbon dioxide from bacterial cultures to detect bacterial contamination in ticks and the transfer to humans, causing Lyme borreliosis. While the standard serological methods to recognize Lyme-disease rely upon the indirect determination of antibodies, the present method enables the direct proof of bacteria both in ticks and in the blood of infected patients, due to the specific hydrogen emission.The most efficient natural antibiotics found were oil of cloves and garlic extract preparations. It was found that the duration of a successful therapy with chemical and natural antibiotics varied but can be perfectly monitored by the gas chromatographic technique.

The technique of static headspace gas chromatography (HS-GC) was applied to monitor the emission of hydrogen and carbon dioxide from bacterial cultures to detect bacterial contamination in ticks and the transfer to humans, causing Lyme borreliosis.This method was also used to compare the efficacy of chemical and natural antibiotics. The most efficient natural antibiotics found were oil of cloves and garlic extract preparations. It was found that the duration of a successful therapy with chemical and natural antibiotics varied but can be perfectly monitored by
the gas chromatographic technique.

Introduction:

Bacteria emit volatile compounds, which are amenable to analysis by static headspace gas chromatography (HS-GC) if cultured in nutrient medium in the septum vials used for HS-GC. The technique of HS-GC has been applied since 1965 in examining the growth of bacteria in milk. Gas chromatography is a well-established technique for automated qualitative and quantitative analysis of volatiles. In addition to the analysis of VOCs released from bacteria, both hydrogen (H2) and carbon dioxide (CO2) are highly informative metabolites. Obligate aerobes produce CO2and water, while facultative anaerobes and microaerophilic anaerobes emit H2 and CO2 together with some other organic compounds, for example ethanol. Tick-borne diseases are difficult to recognize since they often arise late after Borrelia infection and comprise a wide range of various Lyme disease symptoms, such as Lyme arthritis, myalgias, heart abnormalities, chronic fatigue, neuropsychiatric depression and several more. In some areas up to 25% of ticks contain Borrelia bacteria and from these only few percent are also supposed to cause infection. Borrelia are microaerophile and can thrive in an atmosphere with a reduced oxygen concentration compared to air. A simple and easy to handle technique is therefore highly desirable for monitoring the presence of bacteria in the blood of an infected patient and to control the efficacy of an antibiotic therapy. The method of HS-GC proposed in this study may meet this requirement in an excellent way. Lyme-disease, however, may not always be
caused by Borrelia burgdorferi alone, but often by co-infection with subspecies of Borrelia (B. afzellii, B. garinii, B. spielmanii) or by other accompanying facultative bacteria such as Mycoplasma and Yersinia and some more. Our GC-technique can only prove the presence of hydrogen-emitting facultative anaerobic bacteria and not a single species such as Borrelia burgdorferi and for this reason we prefer the term Borreliella as common genus, suggested by S.J. Cutler et al. 

Materials and Methods:

Materials and instrumentation Gas chromatograph GC-AK 11, Aug. Hedinger GmbH & Co KG, Germany; GC-column: 0.8 m x 6 mm polyamide tube, Silica gel 60/80 mesh; headspace vials: 6 ml from Perkin Elmer crimp-capped with PTFE-laminated butyl rubber septa. Nutrient medium: Merck Life Science GmbH, Germany, tryptic soy broth acc EP+ USP 3080r-20p; composition: pancreatic digest of casein, 17g; papaic digest of soya bean meal, 3g; sodium chloride, 5g; dipotassium hydrogen phosphate, 2.5g; glucose monohydrate. Sugar coated tablets with pulverized garlic Kwai©forte 300mg: Cassella-med GmbH & Co.KG,
Germany.

Results:

i) evaluation of the effectiveness of antibiotic treatment and 
ii) supporting the search for effective antimicrobial compounds. Figure 1-Ab shows the comparison of two common antibiotics regarding their antimicrobial activity. Penicillin was found to be ineffective, while doxycycline, the standard antibiotic for Lyme borreliosis worked sufficiently well by complete disappearance of the characteristic hydrogen emission peak (Figure. 1-Ab). In further experiments, three ticks were crushed and incubated in 100 μl of the sample-free nutrient medium, thereby assuming that at least one of them was contaminated. Aliquots of such a homogenous suspension were transferred into the nutrient medium in the vials containing the antibiotics to be examined.

Biography:

Student Research Centre, Überlingen, Obertorstrasse, Germany.