Hospital Acquired Infections with Multiresistant Microorganisms: Can We Escape the Postantibiotic Era?

World wide, millions of patients are affected annually by healthcare-associated infections...


Introduction
World wide, millions of patients are affected annually by healthcare-associated infections (HCAI), impacting up to 8 000 patients in European Hospitals on any given day [1]. The Centers for Disease Control (CDC) estimates that 2 million U.S. patients per year acquire a hospital-related infection. These infections cause 90,000 deaths each year [2,3]. This represents not only a public health risk, but also an economic burden with an average cost of $47,000 per patient to treat. The added cost to hospitals is $ 4.8 billion annually for extended care treatment [4,5]. Bacteria and other microbes evolve in response to their environment and inevitably develop mechanisms to resist being killed by antibiotics and disinfectants [6]. For many decades the problem was manageable as the growth of resistance was slow and the pharmaceutical industry continued to create new antimicrobial agents. The evolution of antibiotic resistance is now occurring at an alarming rate and is outpacing the development of new countermeasures. have been incriminated [7,8]. However, these reasonsalthough important contribute only modestly to the dramatic increased emergence of resistant microorganisms. The emergence of multi resistant microorganisms however developed into a dramatic situation. Biofilm formation and subinhibitory concentrations of disinfectants contribute to this phenomenon [9][10][11]. More than 8000 publications in the international literature describe the resistance of microorganisms against disinfectants and in addition 678 publications describe the cross resistance with antibiotics. Contaminated surfaces contribute substantially to the transmission of hospital pathogens and the spread of multi resitant microorganisms [12]. Disinfection of surfaces with disinfectants is not constructive any more as ample evidence exists for tolerance of microorganisms to sublethal levels of various disinfectants e.g quaternary ammonium compounds (QAC) i.e. benzalkonium chloride as well as chlorhexidine, hexadecylpyridinium and cetrimide [13]. The resistance of QAC based disinfectants to antibiotics is conferred by the resistance determinants qacH and bcrABC. The presence and distribution of these genes have been anticipated to assume a role in the survival and growth of various microorganisms. It has been described that disinfectants (e.g.benzalkonium chloride) even enhance the growth of Listeria monocytogenes in the food industry.
The use of disinfectants -ostensibly intended to remove/kill pathogens on surfaces which are ubiquitously contaminated with microorganisms -is not reliable anymore [14]. Studies have shown that more than one-half the time, microorganisms on these surfaces are not adequately eradicated or surfaces or are recontaminated within minutes. These frequently multiresistant microorganisms are distributed within the hospital by the hands of the personnel to patients. A microbial burden of >8000 CFU on a 100 cm² surface is associated with an incidence of 21 % of an hospital acquired infection. In reality we see 100 -1000 time higher inoculum sizes. [14] Much emphasis has been put therefore on hand disinfection [15]. However, there are also reports of the emergence of alcohol tolerant/insensitive microorganisms e.g. vancomycin resistant enterococci after extensive hand disinfection. This phenomenon has the potential to undermine the effectiveness of alcohol-based disinfectant standard precautions [16][17][18]. This prompted the WHO to publish a warning of an imminent crisis due to the lack of effective antibiotics and requests immediate, coordinate and ambitious measure. An entirely new approach must be instituted [19].
Considering the above information it was mandatory to reassess preventive measures for hospital acquired infections and to curb the dramatic increasing rate of emerging resistant microorganisms.
Antimicrobial coatings hold promise based, in essence, on the application of materials and chemicals with persistent bactericidal or -static properties onto surfaces or in textiles used in the healthcare environments. This belief is based on some preliminary studies involving, for example copper and silver ions, titanium or organosilane, albeit under laboratory conditions. However, the definitive evidence as to their efficacy is still lacking. There are however also several shortcomings of these technologies [20][21][22][23].  Severe/lethal pulmonary toxicity of PHMG was discovered [24].

b) HP Halogenated Phenols:
Responsible for endocrine and neuronal persistent organic pollutant effects, suspected carcinogen [25].

c)
PEI Polyethyleneimine is extremely cytotoxic to eucariontes by two different mechanisms: the disruption of the cell membrane leading to necrotic cell death (immediate) and disruption of the mitochondrial membrane after internalisation leading to apoptosis [26]. This approach is also referred to as biomimetic with respect to the activity of chitosan, a polysaccharide derived from exoskeleton of crustaceans or cell walls of fungi. Chitosan, an aminopolysaccharide biopolymer, has a unique chemical structure with a linear polycation with a high charge density, reactive hydroxyl and amino groups as well as extensive hydrogen bonding. It displays good biocompatibility, physical stability and processability.
Chitosan is known for its antimicrobial activity by penetration of the cell wall and interaction with DNA, inhibiting DNA transcription and ultimately protein synthesis [27]. However, the application of chitosan is only possibly in the form of nanofibers or nanorods which however cannot be stabile anchored on the surface of e.g. a polymer, glass, stainless steel [28]. Nanoparticles of chitosan cannot be incorporated into polymers [29]. Nanotechnologies are subject to approval by the Biocidal product regulation (BPR) of the European

Anti-Adhesive -Hyperhydrophobic Surfaces
Have been designed to reduce the adhesion force between bacteria and a solid surface to enable the easy removal of bacteria before a biofilm layer is formed. Attachment of bacteria or cells starts with an initial adsorption of proteins onto the material surface.
Strategies to prevent protein attachment include superhydrophobic surfaces, often augmented by a hierarchical nanostructure as well as zwitterionic polymers. Such surfaces may suppress health care associated infections by blocking transmission paths involving surfaces, but they will not reduce the number of germs on the surface by killing them. For prevention of recontamination of the hands of the personnel, rapid eradication of microorganisms is mandatory [30]. Quaternary ammonium compounds (QAC) must be excluded from considerations as these products may even enhance the growth of listeria monocytogenes on surfaces and induce cross resistance with antibiotics by induction of efflux pumps. QACs are toxic to a lot of aquatic organisms including fish, daphnids, algae, rotifer and microorganisms employed in wastewater treatment systems. Antibiotic resistance has emerged in microorganisms due to excessive use of QACs in household and industrial applications.
The occurrence of QACs in the environment is correlated with anthropogenic activities, such as wastewater discharge from Wastewater Treatment Plants (WWTPs) or single source polluters. In addition a number of serious adverse events have been described after uptake e.g. coma, convulsions, hypotension and death, hemolysis, allergies, anaphylactic reactions, contact dermatitis. The mechanism of resistance transferred to antibiotics is due to induction of efflux pumps which covers also the majority of antibiotics [31].
In essence the elution of the antimicrobial compounds necessary for incorporation into the metabolism of microorganisms requires at least a moderate water solubility for biocide release and hence a hydrophilic surface. As the antimicrobial agents must be incorporated into the metabolism of microorganisms, one crucial consequence is the high risk of induction of resistance. Nanostructured surfaces were also prepared using electrospun polystyrene nanofibers [33]. With oxygen plasma treatment, a superhydrophilic surface can be generated, which exhibits limited Escherichia coli attachment due to negative zeta potential of -40 mV. After fluorination, a superhydrophobic surface can be obtained, which exhibited self-cleaning ability against bacteria,

Contact-Active Surfaces
where the initially adhering bacteria were effectively removed with subsequent washing [34]. This however is not achievable under clinical conditions.
Mechanisms are dependent on: a. a so-called spacer effect, where the biocidal group is attached to the surface through a polymer chain, allowing the biocide to reach the cytoplasmic membrane of the bacteria and to perforate them [35].
The problem which arises with "spacers" is that the activity of the spacer effect is obliterated by grease, proteins, sweat, pus, blood and tensides. As already indicated the consequences of the use of QACs are the induction of resistance genes in bacteria. The activity of chitosan has been investigated and a poor antimicrobial activity has been found. Last not least these compounds are available only as nanostructures which pose serious problems with acceptance as biocidal products. These technologies although attractive from a theoretical point of view are not applicable for selfsanitizing surfaces for prevention of hospital acquired infections. It has been suggested to combine two functional principles to achieve synergistic effects, e.g. by embedding biocidal substances into antiadhesive surfaces.
Today, the majority of chemical modifications includes hydrogels or polyethylene glycol (PEG) to repel approaching microbes, metals (in particular, silver and copper), antimicrobial peptides (AMPs), quaternary ammonium compounds (QACs), and various nanoparticles [4]. Hydrophobic parts of a surface can act similarly to QACs by deforming the membrane through adhesion. The agents must be insoluble in water-, alcohol-, detergents, acid and alkaline, in addition they must show UV light stability. Antimicrobial agents adjacent to a surfaces have the risk of abrasion with cleaning which is not achievable with the majority of the above mentioned compounds. The topography of a surface can by itself significantly affect its hygienic status, either in a beneficial manner (reducing microbial retention) or otherwise (increasing retention). As such, modifications of surfaces to enhance antimicrobial properties should always take into account the effect of surface wear on subsequent fouling and cleanability. Therefore, efforts should be undertaken to characterize typical wear, assess interactions with the most likely microorganisms in that environment, and define the adverse effects of most appropriate and least damaging cleaning and sanitizing regimes [37].

Anchoring Antimicrobial Agents by Polymer Brushes
The rationale behind the use of polymer brushes is the

a)
AMPs must be incorporated into the metabolism of microorganisms. In contrast to a publication by Gao AMPs must be eluted from the surface in order to effectively penetrate bacterial membranes. Therefore, the activity is limited to a few days if they are not constantly reproduced by epithelial cells.

b)
AMPs are not heat stabile.

c)
AMPs are not easy to be obtained. They could be obtained as Magainins from frog skin with very limited availability [40]. Polycations on a surface are not heat resistant and can´t be extrusion molded. In addition the activity is obscured by many compounds e.g. grease, sweat, pus, blood proteins etc. which are abundantly coating hospital surfaces close to patients [42]. These findings underscore the importance of consistently being aware of the types of strains present in an individual clinic over time as well as monitoring the disinfecting regimens that are most effective against the specific strains. Neglecting this type of monitoring may lead to more serious consequences as bacteria acquire more drug-resistant genes or become otherwise tolerant to disinfectants used in hospital settings. As all of the above described technologies did not meet the requirements for successful eradication of microorganisms from contaminated surfaces it was necessary to search for technologies without the above described limitations.

In Situ Generated Biocides by Use of Transition Metal Oxides
Antimicrobial activity using the concept of Brønsted-Lowry Lewis acids with MoO 3 or similar agents. The anti-microbial effect of acids is well known. Hence the idea was to use acidic surfaces for antimicrobial properties imitating the body´s own defense mechanisms e.g. acid coating of the skin [44][45][46]. Transition

2.
The resulting active substances in addition to acidified water molecules can also be free radicals e.g. oxygen radicals, hydroxyl radicals. There is a synergy on antimicrobial activity by the various mechanisms [48][49][50].

3.
A positive Zeta potential has been observed. A electro positive charge is initiated at the surface which attracts electronegativ charged microorganisms. This results in an almost immediate discharge at the surface and a disruption of the phospholipid bilayer of microorganisms. This is the reason for the rapid bactericidal activity of surfaces [51].

4.
Paramagnetic Ions: documented by EPR spectra. have the least clear described antimicrobial activity but contribute to the synergistic activity of the above described technologies.

Using Transition -Metal Oxides
It is known that MoO 3 reacts with water, e.g. from the ambient air to form H + ions. Therefore, MoO 3 incorporation into polymers e.g. thermoplastic polyurethane, silicone and epoxy resin has been useful for the antimicrobial endowment of surfaces. A similar effective antimicrobial activity has been detected by incorporation of tungsten oxides onto polymers. However, the oxygen saturated tungsten yellow oxide shows a low antimicrobial activity in contrast to the 5 % oxygen deficient tungsten blue oxide. The principle of the technology is shown in Figure 1. and describes the use of MoO 3 for anti-microbial surfaces in thermoplastic polyurethane (TPU).

MoO 3 + 3H2O = 2H 3 O + + MoO 4 2 -
In addition also various free radicals (O2− , hydroxyl radical, •OH) are formed on the surface. Figure 1: Schematic illustration of the technology: MoO 3 (yellow) is embedded in a matrix (blue). At the surface of the compound, germicidal H+ ions will be formed from ambient humidity and various free radicals. Results of electroparamagnetic resonance (EPR) -spectroscopy support these findings.

Zeta potential
Zeta potential is a scientific term for electrokinetic potential

Paramagnetic Ions
The EPR spectra obtained indicate that there are considerable

Experimental Investigations
Since the antimicrobial mechanism of in situ generated biocides is non-specific, there is a broad spectrum of activity including

Molybdenum Trioxide MoO 3
Molybdenum trioxide is available as a light blue/gray powder

2.
Xanthine oxidase catalyses oxidative hydroxylation of purines and pyridines including conversion of hypoxanthine to xanthine and xanthine to uric acid.

4.
Low dietary molybdenum leads to low urinary and serum uric acid concentrations and excessive xanthine excretion.

5.
Molybdenum functions as an electron carrier in those enzymes that catalyse the reduction of nitrogen and nitrate.
Two 13-wk studies were conducted by the national toxicology program (NTP, 1997) in which F-344/N rats and B6C3F1 mice (10/ sex/group) were exposed to molybdenum trioxide for 6.5 hr/d, 5 d/wk at concentrations of 0, 1, 3, 10, 30, or 100 mg/m 3 . All rats and mice survived to the end of the study. Significant increases in liver copper concentrations were observed in female mice exposed to 30 mg/m3 and in male and female mice exposed to 100 mg/m 3 (males: 11.51 µg/g in the 100-mg/m3 exposure group versus 8.19 µg/g in controls; females: 6.51 and 6.98 µ/g in the 30-and 100mg/m 3 dose groups, respectively, versus 5.68 µ/g in controls). The Incidences of hyaline degeneration in the nasal respiratory epithelium in male rats exposed to 30 or 100 mg/m 3 and in all exposed groups of females rats were significantly greater than those of the control groups. Incidences of hyaline degeneration in the nasal olfactory epithelium of all exposed groups of females were also statistically significant. For male mice, the incidences of histiocyte cellular infiltration in all exposed groups were significant.
Incidences of hyaline degeneration of the respiratory epithelium of the nasal cavity in female mice at 100 mg/m 3 were significantly greater than those in the controls (NTP 1997

Tungsten Trioxide WO 3
Tungsten Trioxide has been investigated for in situ generated biocidal activity. Initial experiments with the oxygen saturated Tungsten yellow oxide disclosed limited antimicrobial activity.
Further investigations disclosed that the oxygen deficient tungsten blue oxide WO2.75-2.90 shows antimicrobial activity comparable to Molybdenum trioxide. Tungsten trioxide is a dark blue powder and is available with 5 µm particle size.
Advantage: Tungsten blue oxide is completely water insoluble, the safety data sheet shows no adverse reactions except for necessary individual contact measures. Again this is of relevance to the masterbatch producer as tungsten trioxide is not released from polymers or coatings. Tungsten blue oxide can be used for surfaces in permanent contact with water e.g. pipes. Tungsten blue oxide is a suitable addition to kautschuk e.g. for siderails in escalators.

Zinc Molybdate MoO 4 Zn
The Zinc molybdate is available in 2 µm, 5µm and 8µm particles sizes.
Zinc Molybdate can also be synthesized as 0.2µm particles in fluids in unlimited quantities.
There are 3 possible sources

Additional Remarks
All the above mentioned additives as powder with particle  Figure S1).

Figure S1
: XRD diagrams of the as-prepared MoO 3 based gel and after annealing at various temperatures.

Experimental Investigations
The

Technical Application
Transition metal oxides can be incorporated into various polymers and coatings e.g. liquid silicone, liquid poyurethane, silicium dioxide. It is essential that the surface is hydrophilic i.e.
wettable with a contact angle of 30° or less. This is possible by the addition of various hydrophilising agents e.g. 1% glycerine stearate   Investigations demonstrated excellent antimicrobial efficacy [43].

Toxicologic Investigations and Biocompatibility
Lacquer samples (100 cm 2 containing 0.25 % Zink-Molybdate) have been wiped with a textile 10 000 times with watern, alcohol, detergent. The textile has been investigated by the "spectroturboquant-powder" method. No elution of the additive from the sample has been detected ( Figure 7) Table 1.

Solubility Experiments Of Mo And W In Epoxy Resin And TPU"
One important requirement is the insolubility of Molybdenum There is also a strong antifouling activity.    and mice survived to the end of the study. Significant increases in liver copper concentrations were observed in female mice exposed to 30 mg/m3 and in male and female mice exposed to 100 mg/m 3 (males: 11.51 µg/g in the 100-mg/m 3 exposure group versus 8.19

Investigation of Cytotoxicity
µg/g in controls; females: 6.51 and 6.98 µ/g in the 30-and 100mg/m 3 dose groups, respectively, versus 5.68 µ/g in controls). The increased copper concentrations were not regarded as being an adverse effect relevant for deriving a LOAEL and a NOAEL. No other clinical findings were observed in either rats or mice. Additionally, no significant differences in absolute or relative organ weights, sperm counts, or motility were noted in rats or mice.
In the NTP study (1997), rats (F344/N) and mice (B6C3F1) (50/sex/ dose) were exposed for 6 hr/d, 5 d/wk at concentrations of 0, 10, 30, or 100 mg/m3 molybdenum trioxide for 2 yr experienced a significant exposure-dependent increase in blood Mo concentrations. Male and female rats exposed to 30 or 100 mg/m3 experienced significantly increased incidences of chronic alveolar inflammation. Incidences of hyaline degeneration in the nasal respiratory epithelium in male rats exposed to 30 or 100 mg/m3 and in all exposed groups of females rats were significantly greater than those of the control groups. Incidences of hyaline degeneration in the nasal olfactory epithelium of all exposed groups of females were also statistically significant. For male mice, the incidences of histiocyte cellular infiltration in all exposed groups were significant.
Incidences of hyaline degeneration of the respiratory epithelium of the nasal cavity in female mice at 100 mg/m3 were significantly greater than those in the controls (NTP 1997  titanium is used to form an electrostatic potential at the surface and free radicals such as oxygen radicals to enhance the effectiveness of polyoxometalates. However, titanium is cancerogenic and is therefore not suitable for use on surfaces that many people are exposed to. As a better alternative, other polyoxometalates have been produced using a combination of molybdenum oxide incorporated into the tungsten oxide crystal lattice, which has a stronger zeta potential and also increased formation of free can benefit from the addition of transition metal oxides to the polymer. Selfsanitizing surfaces are also of advantage in nursing homes., hotels and restaurants.
A special application of this technology is dentistry e.g.
coating of dental implants and prostheses as well as the coating of equipment preventing contamination with Hepatitis B and C. The technology is also suitable for antimicrobial paints and coatings for medical equipment e.g. implantable biomaterials, CT scanners, anaesthetic machines, nuclear magnetic resonance imaging, angiography systems, X ray equipment, endoscopes etc.
The technology is also mandatory for germfree surfaces in public transportation, airplanes, trains and trolleys, cockpits of cars e.g.

Patent Protection
The technology of in situ generated biocides is worldwide patent protected. According to the intended use and the specific