Resistance to disinfectants and antibiotics of Pseudomonas spp . and Listeria spp . biofilms on polystyrene and stainless steel

1 Postgraduate Program in Nutrition and Food (PPGNA), Faculty of Nutrition, Federal University of Pelotas (UFPel), Campus Porto, Rua Gomes Carneiro, 1 – Sala 222, CEP 96010-610, Pelotas/RS, Brazil. 2 Graduate curse of Food Technology, Center for Chemical, Pharmaceutical and Food Sciences (CCQFA), Federal University of Pelotas (UFPel), Campus do Capão do Leao, Prédio 31, CEP 96010-900, Pelotas/RS, Brazil. 3 Faculty of Veterinary, Federal University of Pelotas (UFPel), Campus do Capão do Leão, CEP 96010-900, Pelotas/RS, Brazil. 4 Center for Chemical, Pharmaceutical and Food Sciences (CCQFA), Federal University of Pelotas (UFPel), Campus do Capão do Leao, Prédio 31, CEP 96010-900, Pelotas/RS, Brazil.


INTRODUCTION
Increase in consumer demands with regard to the hygiene and sanitary conditions of meat has made producers focus on improvement in microbiological quality and food safety.Meat products are frequently associated with occurrences of food transmitted diseases (FTD) since meat is one of the best medium for the development of bacteria.Bacteria which develop in meat may be connected to deterioration processes or even to the transmission of diseases (Doulgeraki et al., 2012).
Bacteria of the genera Pseudomonas and Listeria may multiply and survive in fridge temperatures and may develop in cold rooms or throughout the cold chain where meat is normally stored (Jay, 2005;Todd and Notermans, 2011).Pseudomonas spp.are particularly deteriorating bacteria and are in the main the cause of the meat´s sensorial alterations, with a consequent decrease in shelf life (Arslan et al., 2011).Within the context of pathogenic bacteria, Listeria monocytogenes causes listeriosis, a serious disease with high lethality rates in risk groups (20-30%) (Lecuit and Leclercq, 2012;EFSA, 2012).
Food industries, especially meat industries, have to face several problems related to cleaning processes and sanitization of utensils and equipments.These problems are often related to the inefficiency of hygiene products and of hygiene processes in the killing or inactivating of microorganisms from the environment, with the subsequent transformation of the sites into focuses of crossed contamination.The above is due to the formation of bacterial biofilms on the equipments and in the production chain.
The formation of biofilms is enhanced in such an environment; it is actually caused by the accumulation of organic and inorganic material used by microorganisms for their fixation on the surface and the subsequent development of biofilms where communities of bacteria establish themselves and resist for long periods (Uhitil et al., 2004;Oliveira et al., 2010).
The term biofilm was created to describe the sessile form of microbial life, characterized by adhesion of microorganisms to biotic or abiotic surfaces, with consequent production of extracellular polymeric substances (Nikolaev et al., 2007;Steenackers et al., 2012).In fact, food industries, especially the processing section, are greatly impaired by biofilms which adhere to various types of surfaces especially stainless steel equipments and utensils (Marques et al., 2007;Sofos and Geornaras, 2010).Further, these bacteria are more resistant to antimicrobial activities and to disinfectant agents, causing deterioration and loss of quality in food and the dissemination of pathogens (Stepanovic et al., 2004;Hamanaka et al., 2012).
Bacterial cells in biofilms may be up to one thousand times more resistant to antibiotics than in their planktonic condition (Ouyang et al., 2012;Sharma et al., 2014).During the last decades, antimicrobial resistance, especially the multi-resistant ones, has been considered a major public health issue worldwide.The excessive and inadequate use of antibiotics may trigger the emergence of resistant bacteria favoring the dissemination of antimicrobial resistant genes in the environment (Filiousis et al., 2009;Domenech et al., 2015).
The relevance of in-depth studies on pathogenic bacteria, with special mention of Pseudomonas spp.and Listeria spp.mainly derived from animal-derived food, such as chicken and buffalo meat, should be underscored.Further, the formation process of biofilms of these bacteria in the food industry should be understood, coupled to their resistance to antibiotics and disinfectant.Preventive and corrective attitudes throughout the food chain to warrant consumers´ health will be adopted.
Current assay aimed at assessing the capacity of Pseudomonas spp.and Listeria spp.originating from chicken and buffalo meat cuts in abattoirs and retail outlets in the southern region of the state of Rio Grande do Sul, Brazil.In the formation of biofilms on polystyrene and stainless steel objects.Considering it biofilm-forming bacteria show greater resistance to antibiotics, like drugs or industrial disinfectant; the resistance to disinfectant agents used in the food industry, and to antibiotics commonly employed in people and animals also will be evaluated.

Bacterial isolates
Current assay employed 69 bacterial isolates.Among the isolates from buffalo meat, there were fourteen isolates identified as Listeria species (1 isolate of L. innocua; 1 isolate of L. rocourtiae and 12 isolates of L. grayi) and twenty-five 25 isolates identified as Pseudomonas genus.Although not all species of Listeria used in this study are pathogenic to man as L. monocytogenes, some are pathogenic to animals and all have similar characteristics and the presence of a species, among chosen in this study, may indicate of the possible presence of L. monocytogenes.Considering the character of deterioration of the genus Pseudomonas in meat, and his capacity to biofilm forming, the genus identification was sufficient for selecting the isolated.They all came from a buffalo abattoir in the southern region of the state of Rio Grande do Sul, Brazil, borrowed from the bacterial bank of the Laboratory of Inspection of Animal derived Products of the Universidade Federal de Pelotas (UFPel).In the case of isolates from chicken meat, five were L. monocytogenes and 25 Pseudomonas spp.derived from the carcasses and meat cuts of chickens from a fowl abattoir and from the retail market in the southern region of Rio Grande do Sul, Brazil.Table 1 shows the isolates´ origin.
The species of Listeria spp.isolated from buffalo meat were confirmed in a previous study with PCR molecular tests with specific primers (data not shown), whereas the species of isolates from chicken meat were confirmed by serological tests undertaken at the Osvaldo Cruz Institution (FIOCRUZ).The genus Pseudomonas spp. was confirmed by biochemical phenotype tests.All isolates were frozen in a Brain and Heart Infusion Broth (BHI, Acumedia ® ) supplemented with glycerol (25%) till use.Listeria spp.isolates were recovered in Tryptone Soy Broth supplemented with 0.6% yeast extract (TSB-YE, Acumedia ® ) whereas Pseudomonas spp.isolates were recovered in a BHI broth.

Evaluation of the biofilms on polystyrene
Bacteria isolates were assessed according to their capacity for biofilm formation on polystyrene microplates following method by  Stepanovic et al. (2007), whereas the maximum volume of each well is 200 µl the plates were washed three times with 200 µL of a sterile saline solution (NaCl 0.9%, pH adjusted to 7.0) to remove all non-adherent cells to the plate.Microplates were inverted on absorbing paper for drying.The samples were then fixed in 150 µL methanol (CH3OH) for 20 min.After this span of time, the methanol was disposed of and the plates were kept upside down during 18 h.Adherent cells were stained with 150 µL violet crystal (0.5%) for 15 min.The stain was then removed under running water and, after drying for 3 min, 150 µL ethanol (CH3CH2OH) (95%) were added.Plates were kept at rest for 30 min and biofilms were counted.The optic density (OD) of the bacterial biofilm was quantified by a microplate reader (ThermoPlate ® ) at 450 nm.
Readings were interpreted following Stepanovic et al. (2007).Mean OD of the samples and of negative control was calculated first; then cut rate (ODc) was calculated as follows: DOc = [average of OD negative control + (3 x standard deviation of negative control)].Final OD rate of tested samples (DOf) was given by ODf = (mean of OD of each sample -DOc).

Assessment of biofilm formation on stainless steel surface
The capacity of biofilm formation on stainless steel surfaces by bacterial isolates was assessed according to method by Rossoni and Gaylarde (2000), with modifications.Stainless steel specimens (AISI 316) measuring 7 cm x 2 cm x 0.1 cm were used.The specimens were immersed in a neutral detergent solution for 1 h; scrubbed manually with a sponge; rinsed with distilled water; sprayed with alcohol 70% and dried at 60°C.They were autoclaved at 121°C for 15 min after sterilization.
Overnight culture were prepared by seeding bacterial isolates separately in 2 mL of BHI broth and incubated at 37°C for 24 h.One milliliter of each culture was added in 40 mL of peptone water 0.85% (Silveira, 2010).Inoculum concentration added to the suspension was standardized with McFarland scale so that the bacterial concentration in 40 mL of peptone water 0.85 % would contain approximately 10 7 CFU/mL.Sterile stainless steel specimens were immersed in the bacterial suspension for 24 h at 25°C.
After immersion, the specimens were washed with 1 mL sterile distilled water to remove all weakly adhering cells.They were then scrubbed by moist swabs and immersed in test tubes with a saline solution 0.1% and homogenized in a tube shaker (Phoenix Luferco ® ) for 3 min (Asséré et al., 2008).Serial decimal dilutions up to 10 -5 were performed for each sample and a 10 µL aliquot of each was seeded in TSA medium (Acumedia ® ) in drops (Silva et al., 2007).Plates were incubated at 37°C for 24 h for CFU counts.The microorganism Staphylococcus epidermidis (ATCC 25923) was the positive control.Biofilm formation on stainless steel specimens was taken into account when counts indicated a number higher than or equal to adhered 10³ CFU/cm 2 , following Wirtanen et al. (1996).

Assessing biofilm removal with disinfectant agents
A modified method by Rossoni and Gaylarde (2000) was employed to assess the removal capacity of biofilm on stainless steel plates with the disinfectant agents organic chlorine and ammonium quaternary at a concentration of 200 parts per million (ppm).Disinfectant agents and their concentration were used due to their wide use in hygiene processes in the food industry.
Induction to biofilm formation on stainless steel specimens was as described above.After the biofilm formation and the last washing, the specimens were immersed separately in flasks with organic chlorine and ammonium quaternary for 10 min.When contact time occurred, the specimens were removed from the disinfectant solution and placed in contact during 3 seconds (s) with a Tween 2% solution to neutralize the ammonium quaternary action.Each specimen was rubbed with moist swabs, followed by immersion in test tubes with a saline solution 0.1% and homogenized with a tube shaker (Phoenix Luferco ® ) for 3 min.Serial decimal dilutions were done for each sample; 10 µL of the suspensions were seeded in Agar TSA by drops (Silva et al., 2007); and plates were incubated at 37°C for 24 h for CFU/cm² counts.Control comprised a specimen of the material with the biofilm immersed in peptone water 0.1%, but not in contact with the disinfectant agent.
The removal of the biofilm from the stainless steel specimens was considered to have occurred when counts were less than or equal to 10² CFU/cm² (APHA, 1992).In this case, statistics tests were realized (analysis of variance and Tukey test at 5%).

The capacity of forming biofilm on polystyrene microplate
Table 2 shows results on the classification of isolates with regard to the formation of biofilms on polystyrene plates, following Stepanovic et al. (2007).Further, 73.7 and 32% were biofilm formers, respectively for isolates Listeria spp.and Pseudomonas spp.All isolates of L. monocytogenes were classified weak biofilm formers.Three L. grayi isolates were classified moderate and four were weak biofilm formers (Table 2).The adhesion of Listeria spp. to the surfaces is greatly facilitated due to its flagella, especially in the initial phases of the biofilm formation (van Houdt and Michiels, 2010).The high number of biofilm-forming Listeria spp.from chicken and buffalo carcasses in the processing demonstrate lack of hygiene in handling, in the sanitization of equipments and utensils and even in the conservation of the product.
Several studies have reported high biofilm formation capacity of L. monocytogenes on polystyrene material (Rodrigues et al., 2010;Kadam et al., 2013) and thus reveal that the material is propitious to colonization by L. monocytogenes biofilms.
One isolate from chicken meat and another from buffalo meat out of the evaluated 50 Pseudomonas spp.isolates were classified as moderate biofilm formers.Nine isolates from chicken meat and 5 from buffalo meat were classified as weak biofilm formers.The above results were corroborated by Ghadaksaz et al. (2015) who registered that 47.1% of the clinical isolates of P. aeruginosa were biofilm former on polystyrene.The low adhesion of Pseudomonas spp.isolates on polystyrene in current analysis occurred because Pseudomonas spp. is a hydrophobic bacterium and tends to adhere on hydrophobic surfaces rather than on hydrophilic ones (Freitas et al., 2010).
Results in current study bring great health concern since the biofilm-forming pathogenic bacteria, such as the L. monocytogenes, and the deterioration-causing ones, such as Pseudomonas spp., are a serious challenge for the food industry since they may cause crossed contamination of products, with subsequent disease transmission and decrease in shelf life (Maia et al., 2009;Giaouris et al., 2014).

The capacity of forming biofilm on stainless steel
All Listeria spp.isolates and 72% of Pseudomonas spp. in current study formed biofilms on stainless steel specimens.Further, 48 and 96% of Pseudomonas spp.isolates respectively retrieved from buffalo and chicken meat formed biofilm on stainless steel.Even if the number of adhered bacterial cells were less than 10³ CFU/cm², there would still be a great risk of microbiological contamination due to microbial concentration (Wirtanen et al., 1996;Oliveira et al., 2010).
Other researchers have shown that, similar to current analysis, bacteria of the genus Listeria have a great ability in adhering to and forming biofilms on the surfaces of stainless steel.The bacterium proves to be a potential risk for the food industry (Moltz et al., 2005;Silva et al., 2008;Berrang et al.,2010;Oliveira et al., 2010;Bonsaglia et al., 2014).
Biofilm formation by Pseudomonas spp.has already been reported in previous studies.Vanhaecke et al. (1990) registered that P. aeruginosa isolates adhered and formed biofilms on stainless steel surfaces within a 30-second contact.Hood and Zottola (1997) showed the formation ability of P. fluorescens biofilm on stainless steel with different culture media.Rossoni and Gaylarde (2000) and Rosado et al. (2006) also demonstrated the capacity of P. fluorescens in forming biofilms on the surface of the same material.When previouslymentioned research works performed in different places, geographically distant one from the other, and results in current study are taken into account, it may be surmised that, regardless of its origin, Pseudomonas spp. is capable of forming biofilms on stainless steel surfaces.The surface adhesion of Pseudomanas spp.may be due to flagella, since these structures give mobility to the bacterium and make it approach the substratum on the surface and, consequently, its adherence (O' Toole and Kolter, 1998).
Results obtained and the use of stainless steel in equipments and on surfaces in food processing demonstrate that L. monocytogenes and Pseudomonas spp.may contaminate food that contact the surfaces if adequate hygiene methods, coupled to adequate disinfectant agents, are not used in the food processing industries.

Biofilm removal by sanitization
Nineteen (19) biofilm formers of isolates of Listeria spp.and 36 isolates of Pseudomonas spp. on stainless steel specimens evaluated in current analysis were assessed for the removal of biofilm by organic chlorine and ammonium quaternary, two common disinfectant agents usually employed in the food industry (Table 3) (Brazil, 1988).
The disinfectant agents should remove pathogenic bacteria and reduce the number of deterioration-causing microorganisms to reasonable levels.For example, 2 CFU/cm² of mesophilic aerobic microorganisms for stainless steel surfaces at the end of the hygienization process (APHA, 1992).Taking into consideration APHA standards, 36.8% of Listeria spp.isolates and 77.7% of Pseudomonas spp.isolates adhered on stainless steel were reduced by organic chlorine.Ammonium quaternary was efficient in removing all Listeria spp.and 91.6% of Pseudomonas spp. on the surface evaluated (Table 3).Ammonium quaternary was more efficient than organic chlorine in case of isolates of Listeria (p = 0.000119), but for Pseudomonas isolates no significant differences between this sanitizers (p=0.238358).Disinfectant agents made from ammonium quaternary have a wide spectrum of activities.In fact, they change their permeability by stimulating glycolysis when in contact with the cell membrane of microorganisms and cause cell exhaustion (Andrade et al., 1996).
Studies that evaluate disinfectant agents in the killing or inactivating of Listeria spp.Biofilms have already been performed.However, only rare reports are extant with regard to isolated of meat cuts and to meat processing industries in south Brazil. Aarnisalo et al. (2007) and Somers and Wong (2004) showed that chlorine-based disinfectant agents were more efficient than ammonium quaternary ones in the elimination of L. monocytogenes adherent to stainless steel.On the other hand, Pan et al. (2006) also analyzed biofilm formation on stainless steel chips and reported the resistance of L. monocytogenes isolates to chlorine and ammonium quaternary.Parikh et al. (2009) assessed the efficiency of three disinfectant agents (lactic acid, sodium hypochloride and ammonium quaternary) inbiofilms composed of L. monocytogenes and reported that all disinfectant agents were efficacious in biofilm decrease.Ammonium quaternary was the most efficient against the developed biofilms.
Several studies analyzed disinfectant agents in the killing or inactivating of Pseudomonas spp.biofilms.Taylor et al. (1999) showed that the treatment of P. aeruginosa with chlorine-based disinfectant agent caused a decrease in biofilm within the space of 5 minutes.Wirtanen et al. (2001) reported that chlorine-based disinfectant agent was efficient in the killing or inactivating of Pseudomonas spp.biofilm from stainless steel surfaces, although tension-active based sanitizers were efficacious in biofilm elimination.Pseudomonas spp.are important bacteria in the food industry since they cause the deterioration of food products and may form biofilms in food processing equipments, albeit with great difficulty in their killing or inactivating due to their resistance to sanitizers (Zhu et al., 2014).
Several researchers have detected high resistance levels to penicillin in L. monocytogenes strains (Harakeh et al., 2009;Fallah et al., 2012), even though concern is greater when L. monocytogenes isolates are resistant to important antibiotics in the treatment of listeriosis.Ampicillin or penicillin with gentamicin is the first choice for the treatment of listeriosis (Charpentier et al., 1999;Conter et al., 2009).
Similar to results in current analysis, the resistance to clindamycin was also reported by Kovacevicet al. ( 2013),    All isolates tested in current analysis are sensitive to chloramphenicol and only one was resistant to gentamicin and ciprofloxacin.Similar results were reported in studies by Doménech et al. (2015) in which all L. monocytogenes isolates from ready-made food were sensitive to the three antibiotics.Gómez et al. (2014) also detected sensitivity to chloramphenicol in all L. monocytogenes isolates and in 99.2% of L. innocua.Kovacevicet al. (2013) reported sensitivity in all Listeria spp.isolates to gentamicin.The high sensitivity of isolates to gentamicin may be due to the fact that it is neither an antimicrobial agent usually used in veterinary therapy nor a growth enhancer in beef cattle (Harakehet al., 2009).
Sensitiveness to trimethoprim-sulfamethoxazole and vancomycin occurred in all isolates in current study.Yan et al. (2010) reported few L. monocytogenes isolates retrieved from food which were resistant to trimethoprimsulfamethoxazole (Sulfazotrim) and vancomycin.However, Kovacevic et al. (2013) and Korsak et al. (2012) reported all isolates as sensitive to vancomycin.Doménech et al. (2015) registered that all L. monocytogenes isolates derived from pork sausages were resistant to trimethoprim-sulfamethoxazole.These results are highly relevant since theantimicrobial agent ranks second in the treatment for listeriosis, especially in patients allergic to penicillin (Pesaventoet al., 2010).According to Harakeh et al. (2009), vancomycin is the last ranking in treatment for infections with listeriosis in humans.
In general terms, L. monocytogenes, retrieved from chicken meat on the retail market, was the only isolate sensitive to all the antibiotics under analysis, although   21% of isolates tested were resistant to two antibiotics and 73.7% were resistant to three to five antibiotics.
Isolates resistant to two or more antibiotics, totally 94.7%, were classified as multi-resistant.In fact, multi-resistance is not restricted to these isolates in southern Brazil since several studies have detected Listeria spp.isolates, multi-resistant to antibiotics, as a worldwide issue (Conter et al., 2009;Yan et al., 2010;Pesavento et al., 2010;Fallah et al., 2012;Goméz et al., 2014).Listeria spp.multi-resistant isolates against antibiotics usually used in the treatment of human listeriosis are a grave issue in public health due to a more difficult therapy especially for people in risk groups, involving elderly people, children, pregnant women and immunocompromised people (Goméz et al., 2014).
Table 5 shows the susceptibility of Pseudomonas spp.isolates to the 12 antibiotics tested, used for treatment of infections mainly caused by P. aeruginosa (Tassios et al., 1998;Jeukens et al., 2014).The highest resistance rate occurred for meropenem, with all isolates derived from chicken meat and 96 % from buffalo meat.There was no resistance in chicken and fish isolates to antibiotics among the P. aeruginosas strains belonging to the carbapenemclass (imipenemand meropenem), evaluated by Maia et al. (2009).In fact, they are used for multiresistant isolates.Results in current analysis are grave since meropenem is an effective antimicrobial agent in the treatment of infections caused by Gram negative bacteria (Gales et al., 2002).
In the case of multi-resistance, 92% of isolates retrieved from chicken meat and 76% of isolates retrieved from buffalo meat were resistant to more than two antibiotics.Multi-resistant increase to antibiotics in Gram negative bacteria and specifically in P. aeruginosa indicate a reduced availability of effective agents for treatments in infections caused by this bacterium.Resistance increase to antibiotics and the potential for global dissemination of resistance genes to pathogen bacteria have become a world health issue for human and veterinarian medicine (Arslan et al., 2011;Sharma et al., 2014).The excessive use of antibiotics in veterinary medicine may be related to pathogens derived from the food chain resistant to antibiotics used by humans (Wang et al., 2007).It is highly important in the context of resistance and multi-resistance to anti-microbial agents to control and monitor the correct employment of these antibiotics in the treatment of people and in veterinary medicine to decrease the transmission of resistance in the food chain.
The testing was performed considering the hypothesis that biofilm-forming bacteria show greater resistance to antimicrobial agents, like drugs, antibiotics or industrial disinfectant.All isolates, who underwent removal test by sanitizers, have formed biofilm on stainless steel.However, not all isolates were resistant antibiotic.Among the Listeria isolates, seven L. gray which were resistant to organic chlorine were also resistant to two types of antibiotics; One L. innocua was resistant the organic chlorine and also to two types of antibiotics; One L. roucotiae which was resistant to organic chlorine was also to five types of antibiotics.Among the three L. monocytogenes, which were resistant to organic chlorine, two of these were resistant to three types of antibiotics and one was sensible to all antibiotics.In the case of Pseudomonas spp isolates, nine isolates were resistant to organic chlorine; eight of these were also resistant at least to two antibiotics.Three isolates, which were resistant to the ammonium quaternary, were also resistant to two types of antibiotics.In this study, it not possible establish a clear relationship positive or negative between the antibiotics and disinfectant resistances verified.

Conclusions
Results demonstrate the importance of control of microbial biofilms in the meat industry since current analysis revealed that isolates of Listeria spp.and Pseudomonas spp.Derived from chicken and buffalo meat were capable of forming biofilms on polystyrene and stainless steel specimens.
The activities of the two disinfectant agents, organic chlorine and ammonium quaternary, were efficient in removing biofilms of Listeria spp.and Pseudomonas spp. on stainless steel specimens.The second agent was more efficient for Listeria spp.So that biofilm risk may be minimized, it is important that the food industry employs control strategies, such as efficient hygiene process that comprises correctly all the stages of cleaning and disinfectant, with recommended products and at the best concentrations for the elimination of microorganisms.
This study identified multi-resistance and resistance to antibiotics in several Listeria spp.and Pseudomonas spp.isolates.

Table 1 .
Origin of Pseudomonas spp.and Listeria spp.isolates from chicken and buffalo meat, in southern Brazil.

Table 2 .
Classification of biofilm formers of Listeria spp.andPseudomonas spp.isolates retrieved from chicken and buffalo meat in southern Brazil.
µg; cefepime 30 µg; ceftazidime 30 µg.Standard cultures at 0.5 concentration in McFarland scale were seeded with a sterile swab in Agar Muller-Hinton (Himedia®) and disks (Multidisco, Laborclin®) impregnated with the above mentioned antibiotics were applied under the surface of the medium.After incubation at 35°C for 24 h, inhibition haloes were measured and interpreted, following CLSI (2005b).

Table 3 .
Efficiency of the disinfectant agents, organic chlorine and ammonium quaternary, in the removal of biofilms formed by Listeria spp.And Pseudomonas spp.retrieved from chicken and buffalo meat, in southern Brazil, on stainless steel specimens.

Table 4 .
Resistance to antibiotics of Listeria spp.isolated retrieved from chicken and buffalo meat, in south Brazil.

Table 5 .
Resistance of Pseudomonas spp.isolates retrieved from chicken and buffalo meat in southern Brazil, to antibiotics.