Introduction to Antimicrobial Polymeric Materials

ALEXANDRA MUÑOZ-BONILLA, MARÍA L. CERRADA AND MARTA FERNÁNDEZ-GARCÍA

Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

This Chapter seeks to bring the readers, in a very brief way, the wonders and the threats that microbes suppose and how human beings fight against them using polymeric materials.

1.1 Short Overview of the World of Micro-Organisms

Microbes are everywhere in the world and their presence constantly affects the environment in which they are growing. The effects of micro-organisms can be beneficial or harmful for their surroundings. Some of them can be positive, sometimes essential, in association with higher forms of organisms (e.g. bacteria and other microbes in the intestines of animals and insects digest nutrients and produce vitamins and growth factors). Moreover, microbes are also used in the manufacture of fermented foods, such as yeasts employed in the fabrication of beer, wine or breads, lactic acid bacteria used to make yogurt, cheese, and other fermented milk products. In addition, microbes are a source in medicine of antibiotics (substances produced by micro-organisms that kill or inhibit other microbes and, then, are used in the treatment of infectious diseases) and vaccines (substances derived from micro-organisms developed to immunise against diseases) for the treatment and prevention of infectious diseases. These advantages come also to biotechnological processes, playing a primary role in recombinant DNA technology and genetic engineering.

Despite these benefits, some microbes cause diseases in animals and plants (pathogens), and they are agents of spoilage and decomposition of foods, textiles and dwellings since nothing lasts forever, and the microbial decomposition of any organic substance will occur with time. Fungi and bacteria are the major microbial agents of decomposition in aerobic environments, while only bacteria can act in anaerobic media. Focusing the attention on the human population, microbial infections still cause around one quarter of all deaths worldwide, especially in undeveloped countries where there are contaminated water or food, unsanitary disposal of human waste, poor personal hygiene, inferior sanitary conditions and lack of access to medical assistance. The magnitude of these infectious diseases (e.g. cholera, dysentery, human immunodeficiency virus infection /acquired immunodeficiency syndrome, malaria, tuberculosis, etc.) is in those countries as significant as they become the first cause of mortality. On the contrary, morbidity and mortality is triggered in developed countries by the increasing incidence of antibiotic-resistant pathogens along with an easily migratory mobility that allow new paths for micro-organisms to run into human hosts to be created. Approximately 25 000 people die each year in the European Union from antibiotic-resistant bacterial infections. For example, Gram-positive Staphylococcus aureus has evolved from penicillin-resistant phenotypes into a methicillin-resistant strain (MRSA), which has become a global epidemic and it is responsible for the main surgical site infections. Countries with the highest rates of resistant infections, such as Greece, Cyprus, Italy, Hungary and Bulgaria, also tend to be the ones with the highest uses of antibiotics. One of the Global Strategy Recommendations dictated by the World Health Organization (WHO) is to make the control of antimicrobial resistance a priority for National Governments and Health Systems. Therefore, new prevention and control strategies are urgently required.

1.1.1 Classification of Micro-Organisms

There are five major groups of micro-organisms: bacteria, algae, fungi, protozoa, and viruses. They are divided into prokaryotic ('before nucleus') and eukaryotic (true nucleus). The former are organisms whose cells lack a cell nucleus (karyon), or any other membrane-bound organelles (only the bacteria and the archaea); the eukaryotic micro-organisms have internal membrane-bound structures, membrane bound nucleus and membrane-bound organelles such as mitochondria, chloroplasts and the Golgi apparatus (algae, protozoa, fungi).

In a simple way, bacteria are prokaryotic and unicellular with a size 1000 times less than the volume of a typical eukaryotic cell, exhibiting different shapes: bacillus (rod), coccus (spherical), spirillum (spiral), vibrio (curved rod).

They are usually classified into two distinct types, Gram-positive and Gram-negative, that differ in the properties of their bacterial cell walls. Gram-positive bacteria are those that are stained dark blue or violet by Gram staining because of the high amount of peptidoglycan in the cell wall. On the contrary, the peptidoglycan layer is thinner in Gram-negative bacteria and is protected by an outer membrane. Consequently, they cannot retain the crystal violet stain, turning in this case reddish or pink by counter-stain (safranin, fuchsine or other stains). In general, Gram-negative bacteria are more resistant against antibiotics compared with Gram-positive ones, because of their outer membrane.

Algae are eukaryotic and unicellular or multicellular; fungi are eukaryotic and unicellular (yeasts) or multicellular (moulds); protozoa (first animals) are eukaryotic and unicellular and viruses are acellular and, then, they are forced to live as intracellular parasites.

1.1.2 Methods of Measuring Microbial Growth

It is of great importance to know the population of micro-organisms and the rates of their growth to inhibit or prevent microbes proliferation. There are numerous techniques of counting microbial growth, measuring either cell mass or cell number, the following are examples:

(a) Dry/wet weight measurement:

This method is a direct approach to determine the net weight of cells. A known volume of culture sample is centrifuged to sediment microorganisms to the bottom of a vessel. The sedimented cells (called a cell pellet) are, then, washed and weighted in case of wet measurements. Dry weight is measured after drying the centrifuged cells. Dry weight is usually about 10–20% of the wet weight, and gives more consistent results and normally is taken as the reference method. These techniques are simple but highly time consuming. In addition, they are not very sensitive and also cannot distinguish between live and dead bacteria.

(b) Absorbance/turbidity:

Absorbance is measured by using a spectrophotometer. Light scattering rises with the increase in cell number. When light is passed through bacterial cell suspension, light is scattered by the cells and transmission decays. At a particular wavelength, light absorbance is proportional to the cell concentration of micro-organisms present in the suspension. This is a nondestructive method that is also very simple, rapid and accurate. Both live and dead cells are, however, able to scatter light and, therefore, both are counted.

(c) Total cell count:

Cell growth is also measured by counting the total cell number of the microbes present in the sample. Total cells (both live and dead) are microscopically counted by using special microscope glass slide or counting chamber, such as, Helber or Petroff–Hausser slides. Typically, this chamber consists of a slide with a grid with etched squares of known area. For example, the surface of the platform is etched with a grid system in the case of Petroff–Hausser chamber. This consists of 25 large squares, each of which is divided into 16 smaller squares. Cells are then counted (normally more than 500) with a phase contrast microscope using a sufficient and appropriate numbers of squares. The number of cells per mL of sample is subsequently calculated from the average cell number per square divided by the volume of a single square. The dilution factors should be taken into account. The main disadvantages of this method are that a high concentration of bacteria is required and the complexity to distinguish between living or dead cells. On the other hand, this is a simple method and does not need any incubation time.

(d) Viable count:

A viable cell is defined as a cell that is able to divide and increase cell numbers. The normal way to perform a viable count is to determine the number of cells in the sample that are capable of forming colonies on a suitable medium. It is assumed that each viable cell will form one colony. Therefore, the viable count is often called the plate count or colony count. This method requires an incubation period of ca. 24 h or longer and can be done in selective and differential. There are two ways of forming plate count: (i) Spread count method: a volume of culture is spread over the surface of an agar plate by using a sterile glass spreader. The plate is incubated to develop colonies. Then, the number of colonies is counted. (ii) Pour plate method: a known volume of the culture is added into sterile Petri dishes. Subsequently, the melted agar medium is poured and gently mixed and, next, incubated. After that, the colonies growing on the surface of the agar are counted. The major shortcoming of this method is the assumption that each colony is generated from a single bacterial cell, thus, sometimes it entails underestimation of the true population. Besides, this technique is time consuming and labour intensive. Despite its disadvantages, the viable plate count is the most frequently used method for measuring cell number because it is very sensitive and allows counting only living bacteria.

(e) Cell-counting instruments:

Coulter counters and flow cytometers are extensively used to count total cells in dilute solutions. Coulter counters are based on electrical impedance. That is, the cells contained in the liquid cause a variation in the electrical impedance that is proportional to the size of the particle. Consequently, the size and the number of cells within a solution can be determined. Flow cytometry is a powerful technique for cell counting that simultaneously measures and, then, analyses physical properties of the cells, such as the light scattering or fluorescence. In this technique, cells are carried in a fluid stream to a laser intercept, thus, thousands of cells pass through a laser beam and the light that emerges from each cell is collected and examined. Flow cytometry can also be used to count organisms to which fluorescent dyes or tags have been attached.

Moreover, there are other methods among all of these common measurements, such as: determination of the amount of a given element, usually nitrogen; acid titration with a pH indicator to quantify acid production; the carbon dioxide formation by using a molecule that fluoresces when the medium becomes slightly more acidic (this gas can be trapped in an inverted Durham tube in a tube of broth); and also ATP measurement using firefly luciferase catalyses light-emitting reaction. However, these methods are more tedious and not often used.

1.1.3 Mechanisms of Action against Micro-Organisms

There are different modes of action against micro-organisms: 1) by affecting their proteins, i.e. denaturation or alteration of their protein structure. This denaturation can be either permanent and, then, the action mechanism is called bactericidal, fungicidal, etc., or temporary if their initial and standard structure can be restored, being then called bacteriostatic, fungistatic, etc. The common mechanisms of denaturation include disruption of hydrogen and disulfide bonds. Another mechanism is 2) by affecting their cell membrane proteins or membrane lipids. Concerning proteins, the mode of action consists of denaturalisation whereas lipids are dissolved, for instance by a surfactant, and their cell membrane turns out to be damaged. Others mechanisms are 3) by affecting the cell-wall formation through blocking its synthesis; 4) by preventing replication, transcription and translation of the nucleic acid structure or 5) by disturbing the metabolism.

1.2 Antimicrobial Polymeric Materials

1.2.1 Brief Introduction

World Health Organization (WHO) has elaborated a catalogue collecting the major concerns about health. The list is the following:

• Alcohol and health.

• Avian influenza A (H5N1).

• Child and adolescent health and development.

• Cholera.

• Environmental and health (household air pollution, outdoor air pollution).

• Expenditures on health (investment on health and on research).

• HIV/AIDS.

• Integrated management of childhood illness (IMCI).

• Influenza.

• Malaria.

• Maternal and reproductive health.

• Meningococcal disease.

• Mortality and burden of disease.

• Neglected tropical disease.

• Noncommunicable diseases (cardiovascular diseases, cancer, diabetes and chronic respiratory diseases).

• Pandemic (H1N1) 2009.

• Poliomyelitis.

• Substance use and substance abuse.

• Tobacco.

• Tuberculosis.

• Violence and injuries.

• Water, sanitation and health.

As can be noticed, many of them are caused by micro-organisms. For example, influenza is a virus and the main strategy to prevent this illness is the vaccination treatment. Cholera is an infection in the small intestine caused by the bacterium Vibrio cholerae. The main symptoms are profuse, watery diarrhoea and vomiting. Transmission occurs primarily by drinking water or eating food that has been contaminated by the faeces of an infected person, including one with no apparent symptoms. In this sense, inadequate access to safe water and sanitation services as well as poor hygiene practices, kills and sickens thousands of children every day, and leads to impoverishment and diminished opportunities for thousands more. Various factors lead to water deterioration, including population growth, rapid urbanisation, agricultural land uses, industrial discharge of chemicals, etc. During recent years, it has been shown that the pharmaceuticals are only partially removed in wastewater treatment plants and have been detected in water bodies. Hospitals contribute in a large extent to the load of wastewater treatment plants, they should be taking into consideration for point-source measures, such as implementing methods to decrease pharmaceuticals or introducing single-use pocket urinals to remove X-ray contrast agents (90–94% are excreted with the urine). In this sense and very recently, Kovalova et al. described a pilot-scale membrane bioreactor operating during one year at a Swiss hospital where 68 target analytes (56 pharmaceuticals, such as antibiotics, antimycotics, antivirals, iodinated X-ray contrast media, anti-inflammatory, cytostatics, etc., 10 metabolites, and 2 corrosion inhibitors) were studied. They make an improvement in the pollutant elimination, almost 90%, when only pharmaceuticals and metabolites are considered without iodinated X-ray contrast media.

Therefore, the cleaning, disinfection and sterilisation of wastewater and/or air is an important target in the antimicrobial policy. However, the contaminants are not only concentrated at outdoor sources; there are also chemical contaminants from indoor supplies. Legionella bacteria and Legionella pneumophila causes legionellosis, a collection of infections that emerged in the second half of the 20th century, and cause a high level of morbidity and mortality in the people exposed. These micro-organisms can be raised in stationary places due to inadequate ventilation, from stationary water, principally in humidifiers or cooling towels, spa pools, spread from air vent systems and/or from water that has been collected on carpets or wood furniture.