Study of Bacterial resistance to biocides

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Bacterial resistance to biocides has been extensively studied since 1950, even if there are several unsolved questions (is resistance due to genetic determinant or an innate property? Is resistance transferable to other individuals and/or species? Which transmission modalities could be involved?). Different possible mechanisms, which could confer lesser sensitivity to biocide, have been described: some of them are related to innate properties of bacterial cells, other instead are focused to population groups. Also innate or acquired tools modulating antimicrobial susceptibility was examined.

One of most exploited theory concerns permeability changes of cell envelope (generally referred as "permeability barrier": this way limits penetration capacity of towards cell inside environment with consequent decreasing of biocidal efficiency (Denyer and Maillard 2002, Lambert 2002, Chapman et al. 2005). Importance of lypopolysaccharides in affecting antimicrobial penetrative ability has been well reported as much as changes in membrane ultrastructure (tattawasart et al. 2000, Broudaki and Hilton 2005).

Other possibly involved mechanism could be enzymatic degradation of biocides such as peroxygens (Valkova et al. 2001, Demple 2001), even if it is still not deeply exploited. Although this biological mechanism could lead to an increase in MIC, this could not involve necessarily a decrease in lethal activity of bactericidal compound (SCENHIR 2009).

Merenghetti et al. (2000) reported that only 7 of 97 Listeria monocytogenes examined strains showed MIC of benzalkonium chloride and cetrimide greater than 7 and 18 μg/ml, respectively, demonstrating that biocide resistance is not homogenously shared among individuals within a certain species. This data is confirmed by Romanova et al. (2002), where quantitatively variable susceptibility to antimicrobial compounds was observed. Best et al. (1990)stated that there are also discrepancies between different species, even if phylogenetically close such as in the case of Listeria innocua and Listeria monocytogenes. Also Soumet et al. (2005) grouped 254 isolates in QAC susceptible (MIC < 3.75 μg/ml) and QAC resistant (MIC > 7.5 μg/ml). Aarestrup et al. (2007) reported for benzalkonium chloride a bimodal distribution, in which 105 isolate could tolerate until 4 μg/ml and nine can withstand until 16 and 32 μg/ml.

Biocidal efficacy is not only concentration-dependent factor (as reported in 2009 SCENHIR Opinion), but it can be affected also by other factor like physiological phase: Adrião et al. (2008) demonstrated that adhered cells showed greater resistance to sodium hypochlorite in comparison with free floating Listeria cells. Lesser susceptibility to sanitizers in biofilm state has been exploited in several scientific papers and different hypothesis have been developed and here briefly discussed (Figure).

Figure: Possible mechanisms of biocide resistance present during growth in biofilm phase (from

First hypothesis concerns environmental conditions during growth in biofilm state: nutrients present in environment are used both by free floating and surface adhered individuals, but in sessile community concentration gradient have been established leading to localized environmental microniches. Deeper the cell is, more starvation-orientered its metabolism is with consequent lesser susceptibility to any antimicrobial molecule;

While planktoi cells are strongly reduced by any adverse condition, cells in biofilms activates mechanism of response to stressful or adverse conditions: this result is achieved sacrificing some individuals of sessile community;

Antimicrobial compounds are not able to penetrate the whole thickness of surface established consortium due to its multilayered structure;

Biocide can reduce significantly number of cells disperse in solution or medium and maybe develop adaption mechanism, whereas, during growth in sessile state, cells are protected by exopolymeric matrix with consequent insurgence of "survivors", which can recolonize microniche after removal of biocidal pressure. Other possible explanation could be found in dormant cells present in deeper layers, which could act as re-colonization nucleus after sanitization.

Biofilm could represent a possible defense against biocidal compounds, even if protection efficacy can be affected by examined compound: Minei et al. (2008) demonstated peroxyacetic acid could reduce significantly Listeria monocytogenes adhered on stainless steel coupons, even if not complete elimantion of inoculums was reached.Sodium hypochlorite seem not to be so effective, as shown by Norwood and Gilmour (2000), where only exposure to 1000 μg/ml (or ppm in this article) free chlorine for 20 min was necessary to produce a 2 log cycle reduction, while 30 s with 10 μg/ml free chlorine exhibit a 8 log reductive effect. This data is confirmed by Hellström et al. (2006), who compared peracetic acid with chlorine in water obtaining 2.1 and 1.2 Log reduction, respectively. Taormina and Beuchat (2002) apply 50-100 mg/ml of cetylpyridinium to have microbial population reduction of more than 5.64 Log.

Benzalkonium chloride is reported by Romanova et al. (2007) to be 1000-fold less effective against Listeria monocytogenes biofilms compared to planktonic analogue. Furthermore Romanova, in same report, state that Listeria monocytogenes biofilms were removed at BC concentrations greater than 10 mg/ml, even if slight re-growth was observed after exposure to 10 mg/ml BC.

Other possible theories, like plasmid- or efflux pump-mediated resistance, have been proposed (Russell 1997, Poole 2007), but not conclusive results regarding L. monocytogenes were obtained (Mereghetti et al. 2000), even if better results were found in other species (e.g. S. aureus, E. coli, Vibrio spp. or Aeromonas spp.).