In heroin injectors, there have been a number of outbreaks caused by spore-forming bacteria, causing serious infections such as anthrax or botulism. These are, most likely, caused by injecting contaminated heroin, and our aim was to develop a filter that efficiently removes these bacteria and is also likely to be acceptable for use by people who inject drugs (i.e. quick, simple and not spoil the hit).
A prototype filter was designed and different filter membranes were tested to assess the volume of liquid retained, filtration time and efficiency of the filter at removing bacterial spores. Binding of active ingredients of heroin to different types of membrane filters was determined using a highly sensitive analytical chemistry technique.
Heroin samples that were tested contained up to 580 bacteria per gramme, with the majority being Bacillus spp., which are spore-forming soil bacteria. To remove these bacteria, a prototype filter was designed to fit insulin-type syringes, which are commonly used by people who inject drugs (PWIDs). Efficient filtration of heroin samples was achieved by combining a prefilter to remove particles and a 0.22 μm filter to remove bacterial spores. The most suitable membrane was polyethersulfone (PES). This membrane had the shortest filtration time while efficiently removing bacterial spores. No or negligible amounts of active ingredients in heroin were retained by the PES membrane.
This study successfully produced a prototype filter designed to filter bacterial spores from heroin samples. Scaled up production could produce an effective harm reduction tool, especially during outbreaks such as occurred in Europe in 2009/10 and 2012.
Many PWIDs filter their heroin before injection, but this only removes particulates to prevent needle blockage and, most likely, reduces small blood vessel damage. Often, PWIDs use homemade pieces of material from cotton wool or cigarettes; these are not sterile and could be a further source of contamination . Some needle and syringe programmes supply filters, but even those commercially available only remove particles (solid materials found in brown heroin, e.g. from poppy straw) that are larger than bacterial spores. The exclusion limit of these filters is, at best, ~10 μm , whereas the average diameter of Bacillus anthracis (the causative agent of anthrax) spores is ~0.8 μm . Whether PWIDs use supplied filters or homemade items, bacterial spores are thus not removed and, if present, could lead to serious and potentially lethal infections. The aforementioned wheel filters are available with a pore size of 0.2 μm and can thus remove bacterial spores, but these only fit syringes with detachable needles and cannot be used with commonly used fixed needle syringes. In addition, these wheel filters retain a significant amount of drug  thus reducing the effect of the drug. Therefore, not all PWIDs would find the use of such filters acceptable.
Bacterial spores of B. subtilis 168  were harvested by first growing overnight in Luria broth (LB; 1% tryptone, 0.5% yeast extract, 1% NaCl), diluting 100-fold in 50 mL Schaeffer sporulation medium  and further growth in a shaking incubator at 37 °C for 3 days. Next, the spores were collected by centrifugation (5000g, 20 min), re-suspended in 10 mL water and centrifuged again, re-suspended in PBS and stored at 4 °C.
Heroin samples were prepared as above and B. subtilis spores were added to a final concentration of ~108 spores/mL. These samples were filtrated, and the flow-through was plated on LB agar plates for enumeration.
In order to remove bacteria (including spores) from heroin, filtration would be the only method that is practical, as the heating of heroin during its preparation does not kill bacterial spores (see below and Ref ). PWIDs would not want drug losses due to filtration; if a significant amount of drug remained in the filter, users would be tempted to extract drug from those filters. A case in point is the use of cotton pellets by heroin users, which in some cases are handled with unwashed hands, stored and re-used to recover any remaining heroin . That is an unsafe practise, as storage of wet filters can lead to growth of microbes and increase risks of infections.
We tested the capability of removing bacterial spores from heroin with the prototype filtration device and a PES membrane. To this purpose, a 0.8 mL heroin sample was spiked with B. subtilis spores (~108 spores), followed by filtration. After this, samples were tested for contamination with B. subtilis spores by plating on agar plates and incubation at 37 °C. No growth was observed after filtration, demonstrating that the filtration device removed bacterial spores efficiently.
Heroin contaminated with bacteria may lead to outbreaks of infectious diseases. The majority of the bacteria identified in brown heroin that we obtained from the local police were Bacillus spp., which are sporulating bacteria that are normally found in soil. It is not surprising that mainly sporulating bacteria were found, as production of heroin from opium involves several steps that require heating and treatment with various chemicals , and only the very resistant bacterial spores could survive these chemicals. None of the bacteria we identified were known to cause disease although it cannot be excluded that they would become pathogenic when injected. Nevertheless, it seems quite likely that only occasional batches with heroin are contaminated with true pathogens such as B. anthracis or Clostridium species. One colony was identified as S. hominis; this is a non-sporulating bacterium that is commonly found on skin and it probably contaminated the heroin after production, e.g. during packaging.
The number of bacteria found in the heroin that we obtained was ~580 bacteria per gramme, which is within the range that was published before . This does not seem a particularly high number, but it should be noted that only a few spores are needed to cause an anthrax infection . Additionally, many PWIDs have poor physical health, which may increase the probability of getting an infection.
Current options for PWIDs to filter heroin include the use of homemade filters from cigarette filters or cotton pellets or purpose-made filters provided by pharmacies or harm reduction programmes (e.g. Sterifilt®, produced by Apothicom) that remove particulates from heroin [7, 8, 10]. These do not remove bacterial contamination [7, 8], but here, we show a prototype filtration device that we developed, a viable option for the removal of bacterial spores and particulates from heroin. Based on both flow properties and low binding of active ingredients of heroin, a prefilter combined with a PES membrane proved to be the most efficient combination. The prefilter removes particulates from the heroin, thereby preventing blocking of the 0.2 μm filter capable of removing bacterial spores. Effectiveness of removal of bacterial spores was tested with spores from B. subtilis, which is a non-pathogenic relative of B. anthracis (and is thus much safer to work with), with spores that are smaller than those of most other sporulating bacteria, including B. anthracis . Thus, a filter designed to work with B. subtilis spores will thus also efficiently remove spores from most bacterial species.
We envisage that after user acceptability testing and suitable production scale-up, our filter could form the cornerstone of infection prevention during anthrax or other spore-forming bacteria outbreak amongst PWID. It could be distributed through Needle and Syringe Programmes (NSPs) at the first detection of an outbreak or ideally supplied continuously. If used for every injection, it would make the use of sterile water (used by some PWIDs) less of a necessity, as any microbes and particles, e.g. from tap water, would also be removed during filtration.
Reinforcement corrosion and the concrete strength reduction are critical problems that resulted from crack creation in concrete. Very expensive and hazardous technologies based on chemical materials have been provided for repairing the cracks. Recently, crack repair using bio-catalysis precipitating bacteria has been developed as a viable and ecofriendly alternative technique. The main target of this study was to select and identify bacterial isolates with high urease activity to use in filling the cracks by the precipitation of CaCO3.
Two endospore-forming and alkali-resistant ureolytic bacteria were combined with concrete to tolerate the mechanical stresses generated by mixing. The two isolates designated as (B1 and B2) were selected and identified as Bacillus wiedmannii strain FSL W8-0169 and Bacillus paramycoides strain MCCC 1A04098, respectively, using 16SrDNA gene sequencing. Both bacterial species completely heal cracks in fully destructed concrete and significant enhancement in compressive strength was illustrated. The calcite filling of cracks and CaCO3 crystals that were screened using a scanning electron microscope may explain the crack healing and the enhancement in concrete strength.
Bacillus wiedmannii strain FSL W8-0169 and Bacillus paramycoides strain MCCC 1A04098 can be inserted with the concrete to improve the compressive strength and the self-healing of cracks. The two ureolytic bacterial strains can be used to protect water buildings from exposure to frequent cracks.
The current research focuses on isolating and identifying endospore-forming, calcite-precipitating, and urease-positive bacteria, as well as determining their suitability for use in concrete. The impact of bacteria on compressive strength and crack healing has also been studied EDX-SEM was used to visually inspect and quantify the calcite precipitation. For the first time, we detected the stalactite deposits in cracks after 90 days water curing of bacterial concrete specimens. 2b1af7f3a8