MUSKI

Evaluation of the efficiency of MUSKI™ as a barrier against microrganisms’ crossing

A) composition of Muski filter

The filter of Muski has the following characteristics:
1.	An outer layer, which is composed by a medical, non woven tissue as a barrier for protecting the inner layers. It has the capacity for filtering substances larger than 2 microns (Figs. 1,2).
2.	An active charcoal layer- 200 g/sq. M. - of which 114 g per sq/m is charcoal, which serves as a filter for smoke, odours, etc. Absorption capability of dust 14.6 g out of 16 g (91,25%) at airflow rate of 10 ft3/min(Figs. 3, 4
3.	A layer that filters substances from microns of size 0,3 or more. Air permeability (cfm/sf) 55. 0,3-0,5 micron 38,6%, 0,5-0,7 micron 68,8%m, 0,7-1,0 micron 78,6%, 1,0. -2,0 micron 83,7% (Figs. 5, 6).
4.	An inner layer witch has the same characteristics of the outer membrane (Figs. 7, 8). Layers 1 and 4 have antiseptic properties because they are also consisted of 0,3% Cetylpyridinium Chloride (CPC) Two different types of Muski (A and B) have been examined. The layers are the above described, but Muski A filter is thinner than Muski B. Consequently breathing is easier with Muski A.
Layers 1 and 4 have antiseptic properties because they are also consisted of 0,3% Cetylpyridinium Chloride (CPC). Two different types of Muski (A and B) have been examined. The layers are the above described, but Muski A filter is thinner than Muski B. Consequently breathing is easier with Muski A.

B) Evaluation of the antiseptic properties of layers 1 and 4

0,1 ml of an 8 hours broth culture (Tryptone Soya Broth) of the different microrganism species tested Staphylococcus cohnii ATCC 35662, Staphylococcus aureus ATCC 6538, Streptococcus pyogenes ATCC 19615, Enterococcus hirae ATCC 10541, Pseudomonas fluorescens ATCC 49838, Escherichia coli ATCC 4157, Salmonella enteritidis ATCC 13076, Serratia marcescens ATCC 8100, Proteus mirabilis ATCC 7002, Bacillus subtilis spores ATCC 6633, Bacillus clausii spores Sonafi-Syntelabo Oto S.p.a.- Milan, Bacillus stearothermophilus spores ATCC 10149, were spread on the surface of Tryptone Soya Agar Petri plate surface using glass spreaders. A fragment of 1cm2 of layers 1 and 4 was put subsequently on the agar inoculated surface and plates were incubated for 24 hours at 36 ± 1 °C for all the micro organisms tested except for the Bacillus stearothermophilus that had to be cultured under the temperature of 56 ± 1 °C . The halo of growth inhibition was then evaluated.

C) Evaluation of the efficiency of Muski as a barrier against microrganisms’ crossing

A bacterial or spore suspension of 1,5 – 7x108 UFC/ml of each of the following micro organisms (Streptococcus pyogenes ATCC 19615, Staphylococcus cohnii ATCC 35662, Serratia marcescens ATCC 8100, Pseudomonas fluorescens 49838, Bacillus clausii spores Sonafi-Syntelabo Oto S.p.a.- Milan, Bacillus stearothermophilus spores ATCC 10149, Bacillus subtilis spores ATCC 6633) was aerosolised by means of a medical device for aerosol therapy. Fragments of Muski filter, previously sterilized by gamma rays, were put into a stainless steel Seitz filter holder, sterilized in steam and plugged in an Erlenmeyer vacuum flask, connected to a vacuum pump, in order to aspirate the microorganisms' aerosol through the Muski. As a control the same described apparatus was used, without the Muski filter. The distance between the aerosol generator and the Seitz filter was about 25 cm. After ten minutes, aerosolisation and vacuum aspirations (0,4m3) were stopped.

In order to evaluate microorganisms' crossing through the apparatus, in the Erlenmeyer flask, 50 ml of a diluent solution (Tryptone natrium chloride) was then introduced in the Erlenmeyer flask, which was then subjected to a mechanical shaker. Both the content of Erlenmeyer and the diluent solution (50 ml), used two times to rinse the Erlenmeyer flask, were filtered through a membrane filtration apparatus. The filtration membrane is transferred to the surface of a Petri dish containing a suitable culture medium according to the species of microorganisms tested. The culture mediums used were:
1.	Pseudomonas isolation agar (Difco) for Pseudomonas fluorescens
2.	Chapman mannitol salt agar (Biomerieux) for *Staphylococcus cohnii*
3.	Streptococcus selective medium (Oxoid) with 5% of defibrinated horse blood for *Streptococcus pyogenes*
4.	Hektoen enteric agar (Biomerieux) for *Serratia marcescens*
5.	Tryptone soya agar (Oxoid) for Bacillus subtilis, Bacillus stearothermophilus and Bacillus clausii. All Petri dishes were incubated at 36±1°C for 24-48 hours and the CFU development was evaluated. For *Bacillus stearothermophilus* the temperature of incubation was 56 ± 1°C.
In order to detect low growing microorganisms, the culture time was extended to a week. No significant differences in the number of CFU were observed between the 24-48 hours culture and the one week culture.
In all experiences an identification with the API system of the developed CFU was done. The developed microorganisms were always the same, which have been previously aerosolised. To evaluate the possible correlation, between the size and the capacity of crossing through the filter of the different tested bacteria and spores, droplets of bacteria or spore suspensions were spread over the surface of a cover glass. The different samples were all fixed for 3 hours at 4°C in a 3% glutaraldehyde solution in a 0.1 M phosphate buffer at pH 7.4 . After several washings in phosphate buffer, the material was dehydrated by passing it through solutions with increasing concentrations of ethyl alcohol, in a propylene oxide solution and finally subjected to critical point drying. The samples were then sputter coated with gold palladium (Edwards sputter coater S150) and examined under a scanning electron microscope (Cambridge Stereoscan S360). The dimensions of 50 bacteria or spores of each species were then measured at SEM.

Results


1.	Antiseptic activity of layers 1 and 4 The halos of inhibition growth for the tested microorganisms are shown in Table 1. At the concentration of 0,3%, *Cetylpyridinium chloride* (which is present in layers 1 and 4 ) demonstrates a high effectiveness against the growth of Bacillus tested species, *B. stearothermophilus* and *B. clausii* and of *Enterococcus hirae, demonstrates a good inhibition against the growth of B. subtilis, Staphylococcus cohnii, Staphylococcus aureus, Streptococcus pyogenes* and results to have moderate effectiveness against the growth of Proteus mirabilis. On the contrary Cetylpyridinium chloride at the concentrations present in layer 1-4, doesn’t seem to have any effects on the growth of *Escherichia coli, Salmonella enteritidis, Serratia marcescens* and Pseudomonas fluorescens .
2.	Efficiency of Muski as a barrier against microorganisms crossing Our results are resumed in table 3 - 4. In our experimental aspiration apparatus the presence of Muski filter strongly reduced the crossing of microorganisms and spores aerosolised in the air. In the case of aerosolization of vegetative forms of microorganisms the percentage (%) of reduction varies between 98,30% and 100%, whereas by aerosolising *Bacillus* spores results a likely lower percentage of reduction (from 94,25% to 99,12%). No evident correlation seems to exist between microorganisms’ or spores’ size and the capacity of crossing the filter of Muski (compare Table 2 with Table 3 - 4 and Figs. 9-15).

It also seems to exist no difference between the capacity of penetration through the Muski filter of spherical micro organisms (*Staphylococcus cohnii* and *Streptococcus pyogenes) and rod shaped bacteria like Pseudomonas fluorescens* and *Serratia marcescens*. It seems also very difficult to correlate the sensibility against the antibiotic (Cetylpyridinium Chloride) of different microorganisms tested with their capacity of crossing alive the different layers of the Muski filter. We consider that the good barrier effect of Muski against microorganisms is correlated to both the structure of different layers and the antibiotic, which is present in the outer and in the inner layers of the Muski filter. In conclusion our results demonstrate that Muski filter is a very efficient barrier against bacteria or spores’ crossing.

Table 1
Halos of growth inhibition produced by Muski layers 1 and 4.

Type of microrganism	Layer 1	Layer 4
*Escherichia coli*	0mm	0mm
*Salmonella enteritidis*	0mm	0mm
*Serratia marcescens*	0mm	0mm
*Proteus mirabilis*	1mm	1mm
*Pseudomonas fluorescens*	0mm	0mm
*Staphylococcus cohnii*	2mm	2mm
*Staphylococcus aureus*	3mm	2.9mm
*Streptococcus pyogenes*	2mm	2mm
*Enterococcus hirae*	10mm	11mm
*Bacillus subtilis*	2mm	2mm
*Bacillus clausii*	8mm	8mm
*Bacillus stearothermophilus*	8mm	9mm

TABLE 2
Size (mean and standard deviation) of different microrganisms tested; diameter for spherical bacteria, length and breadth for rod shape bacteria or spores.

Type of microrganism	Size in Micron
*Staphylococcus cohnii*	0.70 ± 0.08
*Streptococcus pyogenes*	0.80 ± 0.16
*Pseudomonas fluorescens*	1.50 ± 0.49 x 0.51 ± 0.03
*Serratia marcescens*	1.10 ± 0.60 x 0.37 ± 0.04
*Bacillus stearothermophilus spores*	0.86 ± 0.13 x 0.53 ± 0.04
*Bacillus clausii spores*	1.01 ± 0.13 x 0.57 ± 0.05
*Bacillus subtilis spores*	1.25 ± 0.15 x 0.58 ± 0.07

TABLE 3
Number of developed CFU, in the filtration apparatus, and % reduction with or without Muski filters after aerosolization of different bacteria.

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
*Staphilococcus cohnii*	with MUSKI	0	A	100%
	without MUSKI	516
	with MUSKI	0	B	100%
	without MUSKI	8000
	with MUSKI	15	A	99.770%
	without MUSKI	6300

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
*Streptococcus pyogenes*	with MUSKI	0	B	100%
	without MUSKI	233
	with MUSKI	0	B	100%
	without MUSKI	840
	with MUSKI	2	A	98.88%
	without MUSKI	1650

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
*Serratia marcescens*	with MUSKI	5	A	99.4%
	without MUSKI	8004
	with MUSKI	0	B	100%
	without MUSKI	304
	with MUSKI	2	A	99.95%
	without MUSKI	4000

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
*Pseudomonas fluorescens*	with MUSKI	3	A	98.3%
	without MUSKI	176
	with MUSKI	9	B	98.33%
	without MUSKI	538
	with MUSKI	3	A	99,65 %
	without MUSKI	850

TABLE 4
Number of developed CFU, in the filtration apparatus, and % reduction with or without Muski filters after aerosolization of different bacteria

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
*Bacillus clausii* (spores)	with MUSKI	95	A	97.93%
	without MUSKI	4440
	with MUSKI	95	B	97.64%
	without MUSKI	4025
	with MUSKI	17	B	97,50 %
	without MUSKI	680
	with MUSKI	46	B	96,00 %
	without MUSKI	1148

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
Bacillus stearothermophilus (spores)	with MUSKI	3	A	99.12%
	without MUSKI	340
	with MUSKI	9	B	97.84%
	without MUSKI	411

Test Microrganism	N^{o of CFU}		Type of Muski	Reduction with MUSKI
Bacillus subtilis (spores)	with MUSKI	30	B	94.25%
	without MUSKI	523
	with MUSKI	4	B	97.45%
	without MUSKI	157
	with MUSKI	18	A	96.37 %
	without MUSKI	496

Pictures

Figs 1-2 - Outer layer of the Muski filter.
Figs 3-4 - Active charcoal layer of Muski filter. In fig 3 some micro fibres of the outer layer are sticking to the charcoal layer.
Figs 5-6 - Intermediate layer of Muski filter.
Figs 7-8 - Inner layer of Muski filter.
Fig 9 - Scanning electron micrograph of Staphilococcus cohni.
Fig 10 - Scanning electron micrograph of Streptococcus pyogenes.
Fig 11 - Scanning electron micrograph of Serratia marcescens.
Fig 12 - Scanning electron micrograph of Pseudomonas fluorescens.
Fig 13 - Scanning electron micrograph of Bacillus stearothermophilus spores.
Fig 14 - Scanning electron micrograph of Bacillus clausii spores.
Fig 15 - Scanning electron micrograph of Bacillus subtilis spores

Outer layer of the Muski filter.