Cytotoxicity and inflammatory potential of two Pseudomonas mosselii strains isolated from clinical samples of hospitalized patients
© Leneveu-Jenvrin et al.; licensee BioMed Central Ltd. 2013
Received: 25 September 2012
Accepted: 24 May 2013
Published: 29 May 2013
The genus Pseudomonas includes a heterogeneous set of microorganisms that can be isolated from many different niches and nearly 100 different strains have been described. The best characterized bacterium is Pseudomonas aeruginosa which is the primary agent of opportunistic infection in humans, causing both acute and chronic infections. Other species like fluorescens, putida or mosselii have been sporadically isolated from hospitalized patients but their association with the pathology often remains unclear.
This study focuses on the cytotoxicity and inflammatory potential of two strains of Pseudomonas mosselii (ATCC BAA-99 and MFY161) that were recently isolated from clinical samples of hospitalized patients. The behavior of these bacteria was compared to that of the well-known opportunistic pathogen P. aeruginosa PAO1. We found that P. mosselii ATCC BAA-99 and MFY161 are cytotoxic towards Caco-2/TC7 cells, have low invasive capacity, induce secretion of human β-defensin 2 (HBD-2), alter the epithelial permeability of differentiated cells and damage the F-actin cytoskeleton.
These data bring new insights into P. mosselii virulence, since this bacterium has often been neglected due to its rare occurrence in hospital.
KeywordsPseudomonas mosselii Cytotoxicity Inflammation F-actin cytoskeleton damage
The genus Pseudomonas is one of the most diverse and ecologically significant bacterial groups on the planet. The large size and the plasticity of their genome explain at least partly their ability to cope with different forms of stresses (physical, chemical or antimicrobial agents) resulting in their widespread distribution . The genus Pseudomonas includes more than 100 species, a number that is increasing in time . Nearly each year, a new species is indeed discovered, like P. duriflava, P. batumici or P. litoralis for example, isolated from a desert soil , the Caucasus Black sea coast  or from Mediterranean seawater , respectively. Due to its heterogeneity, the genus Pseudomonas has undergone numerous taxonomic changes depending on the criteria employed for their definition and delineation: phenotypic, physiologic or metabolic characteristics, siderotyping, phylogeny based on 16S rRNA and/or “housekeeping” genes, analysis of 16S-23S rRNA intergenic spacers (ITS) or the use of functional and ecological genetic markers such as oprF, oprD or gacA[2, 6–8].
P. aeruginosa is by far the most studied species in the genus Pseudomonas. It is an opportunistic pathogen that provokes nosocomial infection and causes severe acute and chronic infections either in healthy or in immunocompromised individuals . Other Pseudomonas species have been suspected in human infections . For example, the very common environmental contaminant P. fluorescens has also been associated to various clinical cases [10–14]. This bacterium may particularly colonize the airways, the urinary tract and blood of immunocompromised patients. Recently, some P. fluorescens strains were found to behave as human pathogens, since they have a high hemolytic activity and dispose of a complete type three secretion system arsenal [15–18].
P. mosselii is a novel species, which has been characterized in 2002 . It has been linked to P. putida clinical strains using 16SrDNA, oprF and oprD as markers for phylogeny-based studies [7, 8]. In 2009, McLellan and Partridge  presented a case of prosthetic valve endocarditis caused by P. mosselii. These authors proposed that P. mosselii should be regarded as a potential pathogen.
In a previous study, we have found that P. mosselii strains were able to adhere and to display a necrotic potential on rat glial cells . To get further insights into P. mosselii virulence, we investigate in the present work the cytotoxicity and proinflammatory effects of two clinical strains of P. mosselii (ATCC BAA-99 and MFY161) on Caco2/TC7 cells, the transepithelial permeability of Caco2/TC7 monolayers and the actin network. The behavior of these bacteria was compared to that of the well-known opportunistic pathogen P. aeruginosa PAO1.
Bacterial invasion assay
Quantification of IL-6, IL-8 and HBD-2 secretion
The human β-defensin-2 (HBD-2) was also quantified in Caco-2/TC7 cells supernatant. The results show that the two strains of P. mosselii were able to induce HBD-2 secretion by Caco-2/TC7 cells (Figure 3C). Infection with P. mosselii ATCC BAA-99 and MFY161 strains led to a major increase of HBD-2 production by Caco-2/TC7 with 125 +/−26 pg.mL-1 and 136 +/−31 pg.mL-1, respectively, compared to the 4 +/−2 pg.mL-1 basal secretion of HBD-2 in uninfected cells. The induction of HBD-2 by the two P. mosselii strains was almost similar to that obtained with P. aeruginosa PAO1 (165 +/−14 pg.mL-1).
Transepithelial electrical resistance measurements
P. mosselii was formally described as a novel species in 2002 through a polyphasic taxonomic approach including 16SrDNA phylogeny, numerical analysis, DNA–DNA hybridization, thermal stability of DNA–DNA hybrids and siderophore-typing methodology . The several strains of P. mosselii described to date were isolated in hospital and some have been suggested as emerging human pathogens [19–21]. Our study aimed at investigating the virulence potential of two of these strains, namely ATCC BAA-99 and MFY161, belonging to the same cluster strongly related to the hospital-isolated P. putida on the basis of both oprD or oprF-linked phylogenies . Although P. putida species is mostly known for its huge capacity in degradation of numerous carbon sources , some clinical strains have emerged, causing infections in immunosuppressed hosts and patients with invasive medical devices. More recently, P. putida has been involved in war wound infection, and should be considered as a potential human pathogen, for a review see Carpenter et al. .
In the present study, we further investigated the cytotoxicity of P. mosselii ATCC BAA-99 and MFY161 strains, and show that they provoked the lysis of the intestinal epithelial cells Caco-2/TC7, with a major damage obtained after infection with P. mosselii MFY161. The cytotoxic levels were lower compared to the well-known opportunistic pathogen P. aeruginosa PAO1 but almost similar to those observed for P. mosselii strains on rat glial cells , and for the clinical strain P. fluorescens MFN1032 on Caco-2/TC7 cells . The gentamicin exclusion test showed that P. mosselii ATCC BAA-99 and MFY161 can enter Caco-2/TC7 cells. The invasion capacity of the two P. mosselii strains studied was similar and lower than that of the pathogen P. aeruginosa PAO1.
The bacterial proinflammatory effect of P. mosselii ATCC BAA-99 and MFY161 was then assessed by measuring the secretion of IL-6 and IL-8 cytokines in Caco-2/TC7 after 24 h of infection. The results showed that the two strains did not induce the production of these proinflammatory cytokines. We hypothesize that this may serve as a strategy for P. mosselii to escape the immune system. However, P. mosselii ATCC BAA-99 and MFY161were found to strongly increase the secretion of HBD-2. Human beta-defensins are known to play a key role in host defense. In fact, in addition to their potent antimicrobial properties against commensal and pathogenic bacteria , beta-defensins were demonstrated to function as multieffector molecules capable of enhancing host defense by recruiting various innate as well as adaptive immune cells to the site of infection. Nevertheless, some pathogens can be resistant to HBD-2  and surprisingly can induce and divert HBD-2 secretion in intestinal epithelial cells to enhance its capacity of virulence .
The effect of P. mosselii ATCC BAA-99 and MFY161 on epithelial permeability was then evaluated by measuring the TER across differentiated Caco-2/TC7 monolayers. The F-actin cytoskeleton was stained with Alexa-488 phalloïdin and examined using a confocal laser scanning microscope. We observed that the TER of the monolayers exposed to the bacteria was significantly decreased and that the F-actin cytoskeleton was completely broken. Similar results of TER decrease and F-actin disruption were previously observed with many pathogens including Salmonella typhimurium, P. aeruginosa and Escherichia coli[28–30].
Infections caused by multidrug-resistant (MDR) Gram-negative bacilli have become a growing challenge in hospital . In a recent study, Giani et al.  suggested that unusual human opportunistic pathogen like P. mosselii may probably play a role as shuttles for acquired metallo-β-lactamases resistance thus an antibiogram was made for P. mosselii ATCC BAA-99 and MFY161 (see Additional file 1: Table S1). We found that the two strains were resistant towards 6 of the 16 antibiotics tested including the ticarcillin beta-lactam, which could support the above hypothesis.
In conclusion, our study demonstrates that P. mosselii ATCC BAA-99 and MFY161 are cytotoxic towards Caco-2/TC7 cells, have low invasive capacity, induce secretion of human β-defensin 2 (HBD-2), alter the epithelial permeability of differentiated cells and damage the F-actin cytoskeleton. These strains are less virulent than P. aeruginosa PAO1, but their behavior resembles that of cytotoxic strains of P. fluorescens[17, 18] and by thus may be considered as potential emerging human pathogen.
P. mosselii ATCC BAA-99 is a clinical strain isolated from tracheal aspirate of a patient suffering from pulmonary infections . P. mosselii MFY161 was collected from urine of a patient suffering from alcoholic hepatitis in Charles Nicolle hospital (Rouen, France), and characterized by 16SrDNA, oprF and oprD sequencing [7, 8], and siderotyping . P. aeruginosa PAO1 was obtained from an international collection. All the strains were routinely cultivated under vigorous shaking, in ordinary nutrient broth (Merk, Darmstadt, Germany), at their optimal growth temperature, 30°C for P. mosselii ATCC BAA-99 and MFY161, 37°C for P. aeruginosa PAO1.
Cell line and culture
Caco-2/TC7 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) supplemented with 15% of heat-inactived fetal calf serum, 2 mM of L-glutamine, 100 U.mL-1 each of penicillin and streptomycin and 1% of non-essential amino acids. For the experimental assays, the cells were seeded at a density of 105 cells.cm-2 in 24-wells tissue culture plates, or on inserts (6.4 mm diameter, 3 μm pore size, Falcon) to obtain fully differentiated cells. The cells were cultured at 37°C in 5% CO2-95% air atmosphere and the medium was changed daily.
Bacteria of overnight culture were harvested by centrifugation (5000 g, 5 min, 20°C) and resuspended at a density of 108 colony forming unit (CFU).mL-1 in cell culture medium without serum and antibiotics. Caco-2/TC7 cells grown on 24-wells culture plates or inserts were washed twice with fresh culture medium and the bacterial suspensions were applied to the cell surface at a concentration of 108 CFU.cm-2, resulting to a multiplicity of infection (MOI) of 100. Infected cells were then incubated at 37°C in 5% CO2-95% air during 24 h for all experiments, excepted 4 h of infection for the invasion test. Each assay was conducted in triplicate in independent experiments (successive passages of Caco-2/TC7 cells).
Cytotoxicity assay was performed on confluent Caco-2/TC7 grown in 24-wells culture plates. After 24 h of infection, the supernatants from Caco-2/TC7 monolayers were collected and the concentration of lactate dehydrogenase (LDH), a cytoplasmic enzyme released upon cell death, was determined using an enzymatic assay (Cytotox 96 Promega, Charbonnieres, France) as previously described . Caco-2/TC7 cells exposed to Triton ×100 (0.9%) were used as a control of total LDH release (100% dead cells).
Bacterial invasion assay
After 4 h of infection, Caco-2/TC7 monolayers were washed with phosphate-buffered saline (PBS). Adherent bacteria were killed by incubation for 1 h with 300 μg.mL-1 gentamycin, an antibiotic that does not cross the cytoplasmic membrane of eukaryotic cells and then only kills bacteria not internalized in cells. Caco-2/TC7 monolayers were washed 3 times with PBS to remove the antibiotic and dead bacteria. The cells were then lysed by incubation for 15 min with 0.5% Triton ×100 to release the intracellular bacteria and the lysates were plated onto nutrient agar to determine the number of internalized bacteria.
Quantification of IL-6, IL-8 and HBD-2
After 24 h of infection with the bacterial suspensions, the levels of IL-6 and IL-8 cytokines were measured in Caco-2/TC7 cells supernatant using ELISA Quantikine kits (R&D systems). The human β-defensin-2 (HBD-2) was quantified using the Defensin 2, beta (Human) - ELISA Kit (Phoenix Pharmaceuticals inc). These assays were conducted according to the manufacturer’s protocols.
Transepithelial electrical resistance measurements
Caco-2/TC7 cells grown on inserts were used at 21 days post-confluence (fully differentiated cells) and the transepithelial electrical resistance (TER) of the monolayers infected or not with the bacterial strains was measured during 24 h using the Millicell Electrical Resistance System (Millipore Corp, Bedford, MA). TER values are expressed as percentages of the pre-infection level of the TER (baseline) measured for each individual cell monolayer in the inserts.
Fully differentiated Caco-2/TC7 monolayers were exposed to the bacterial strains for 24 h. At the end of the experiment, the cells were washed with PBS, fixed for 10 min in 3.7% paraformaldehyde and permeabilized for 5 min with 0.1% Triton ×100 at room temperature. The cells were then incubated with 1% bovine serum albumin in PBS for 10 min and the apical F-actin cytoskeleton was stained with Alexa-488 phalloïdin (1U/insert) for 45 min at room temperature. Following three washes in PBS, cell monolayers were examined using a confocal laser scanning microscope (Zeiss, LSM710).
All experiments were conducted independently at least three times. The results are expressed as means +/− SEM and statistical significance were performed by Student’s t-test.
Charlène Leneveu-Jenvrin is a recipient of a doctoral fellowship from the region Haute-Normandie (GRR-SSE). This study was supported by grants from the Conseil Général de l’Eure, the Grand Evreux Agglomération and FEDER funds. LMSM is a member and is supported by the world’s leading centre Cosmetic Valley.
- Spiers AJ, Buckling A, Rainey PB: The causes of Pseudomonas diversity. Microbiology. 2000, 10: 2345-2350.View ArticleGoogle Scholar
- Peix A, Ramirez-Bahena MH, Velazquez E: Historical evolution and current status of the taxonomy of genus Pseudomonas. Infect Genet Evol. 2009, 9: 1132-1147. 10.1016/j.meegid.2009.08.001.PubMedView ArticleGoogle Scholar
- Liu R, Liu H, Feng H, Wang X, Zhang CX, Zhang KY, Lai R: Pseudomonas duriflava sp. nov., isolated from a desert soil. Int J Syst Evol Microbiol. 2008, 58: 1404-1408. 10.1099/ijs.0.65716-0.PubMedView ArticleGoogle Scholar
- Kiprianova EA, Klochko VV, Zelena LB, Churkina LN, Avdeeva LV: Pseudomonas batumici sp. nov., the antibiotic-producing bacteria isolated from soil of the Caucasus Black Sea coast. Mikrobiol Z. 2011, 73: 3-8.PubMedGoogle Scholar
- Pascual J, Lucena T, Ruvira MA, Giordano A, Gambacorta A, Garay E, Arahal DR, Pujalte MJ, Macian MC: Pseudomonas litoralis sp. nov., isolated from Mediterranean seawater. Int J Syst Evol Microbiol. 2012, 62: 438-444. 10.1099/ijs.0.029447-0.PubMedView ArticleGoogle Scholar
- Costa R, Gomes NC, Krogerrecklenfort E, Opelt K, Berg G, Smalla K: Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses. Environ Microbiol. 2007, 9: 2260-2273. 10.1111/j.1462-2920.2007.01340.x.PubMedView ArticleGoogle Scholar
- Bodilis J, Calbrix R, Guerillon J, Merieau A, Pawlak B, Orange N, Barray S: Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences. Syst Appl Microbiol. 2004, 27: 93-108. 10.1078/0723-2020-00253.PubMedView ArticleGoogle Scholar
- Chevalier S, Bodilis J, Jaouen T, Barray S, Feuilloley MG, Orange N: Sequence diversity of the OprD protein of environmental Pseudomonas strains. Environ Microbiol. 2007, 9: 824-835. 10.1111/j.1462-2920.2006.01191.x.PubMedView ArticleGoogle Scholar
- Obritsch MD, Fish DN, MacLaren R, Jung R: Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options. Pharmacotherapy. 2005, 25: 1353-1364. 10.1592/phco.2005.25.10.1353.PubMedView ArticleGoogle Scholar
- Wei B, Huang T, Dalwadi H, Sutton CL, Bruckner D, Braun J: Pseudomonas fluorescens encodes the Crohn’s disease-associated I2 sequence and T-cell superantigen. Infect Immun. 2002, 70: 6567-6575. 10.1128/IAI.70.12.6567-6575.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Sutton CL, Kim J, Yamane A, Dalwadi H, Wei B, Landers C, Targan SR, Braun J: Identification of a novel bacterial sequence associated with Crohn’s disease. Gastroenterology. 2000, 119: 23-31. 10.1053/gast.2000.8519.PubMedView ArticleGoogle Scholar
- Dalwadi H, Wei B, Kronenberg M, Sutton CL, Braun J: The Crohn’s disease-associated bacterial protein I2 is a novel enteric t cell superantigen. Immunity. 2001, 15: 149-158. 10.1016/S1074-7613(01)00164-9.PubMedView ArticleGoogle Scholar
- Feuilloley MGJ, Mezghani-Abdelmoula S, Picot L, Lesouhaitier O, Merieau A, Guerillon J, Boujedaini N, Cazin L, Orange N: Involvement of Pseudomonas and related species in central nervous system infections. Res. Dev. Microbiol. 2002, 7: 55-71.Google Scholar
- Bernstein DI, Lummus ZL, Santilli G, Siskosky J, Bernstein IL: Machine operator’s lung. A hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols. Chest. 1995, 108: 636-641. 10.1378/chest.108.3.636.PubMedView ArticleGoogle Scholar
- Hsueh PR, Teng LJ, Pan HJ, Chen YC, Sun CC, Ho SW, Luh KT: Outbreak of Pseudomonas fluorescens bacteremia among oncology patients. J Clin Microbiol. 1998, 36: 2914-2917.PubMedPubMed CentralGoogle Scholar
- Rossignol G, Merieau A, Guerillon J, Veron W, Lesouhaitier O, Feuilloley MG, Orange N: Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens. BMC Microbiol. 2008, 8: 189-10.1186/1471-2180-8-189.PubMedPubMed CentralView ArticleGoogle Scholar
- Madi A, Lakhdari O, Blottiere HM, Guyard-Nicodeme M, Le Roux K, Groboillot A, Svinareff P, Dore J, Orange N, Feuilloley MG, Connil N: The clinical Pseudomonas fluorescens MFN1032 strain exerts a cytotoxic effect on epithelial intestinal cells and induces Interleukin-8 via the AP-1 signaling pathway. BMC Microbiol. 2010, 10: 215-10.1186/1471-2180-10-215.PubMedPubMed CentralView ArticleGoogle Scholar
- Madi A, Svinareff P, Orange N, Feuilloley MG, Connil N: Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells. Gut Pathog. 2010, 2: 16-10.1186/1757-4749-2-16.PubMedPubMed CentralView ArticleGoogle Scholar
- Dabboussi F, Hamze M, Singer E, Geoffroy V, Meyer JM, Izard D: Pseudomonas mosselii sp. nov., a novel species isolated from clinical specimens. Int J Syst Evol Microbiol. 2002, 52: 363-376.PubMedView ArticleGoogle Scholar
- McLellan E, Partridge D: Prosthetic valve endocarditis caused by Pseudomonas mosselii. J Med Microbiol. 2009, 58: 144-145. 10.1099/jmm.0.005553-0.PubMedView ArticleGoogle Scholar
- Chapalain A, Rossignol G, Lesouhaitier O, Merieau A, Gruffaz C, Guerillon J, Meyer JM, Orange N, Feuilloley MG: Comparative study of 7 fluorescent pseudomonad clinical isolates. Can J Microbiol. 2008, 54: 19-27. 10.1139/W07-110.PubMedView ArticleGoogle Scholar
- Bodilis J, Hedde M, Orange N, Barray S: OprF polymorphism as a marker of ecological niche in Pseudomonas. Environ Microbiol. 2006, 8: 1544-1551. 10.1111/j.1462-2920.2006.01045.x.PubMedView ArticleGoogle Scholar
- Poblete-Castro I, Escapa IF, Jager C, Puchalka J, Lam CM, Schomburg D, Prieto MA, Martins dos Santos VA: The metabolic response of Pseudomonas putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. Microb Cell Fact. 2012, 11: 34-10.1186/1475-2859-11-34.PubMedPubMed CentralView ArticleGoogle Scholar
- Carpenter RJ, Hartzell JD, Forsberg JA, Babel BS, Ganesan A: Pseudomonas putida war wound infection in a US Marine: a case report and review of the literature. J Infect. 2008, 56: 234-240. 10.1016/j.jinf.2008.01.004.PubMedView ArticleGoogle Scholar
- Eckmann L: Defence molecules in intestinal innate immunity against bacterial infections. Curr Opin Gastroenterol. 2005, 21: 147-151. 10.1097/01.mog.0000153311.97832.8c.PubMedView ArticleGoogle Scholar
- Moranta D, Regueiro V, March C, Llobet E, Margareto J, Larrarte E, Garmendia J, Bengoechea JA: Klebsiella pneumoniae capsule polysaccharide impedes the expression of beta-defensins by airway epithelial cells. Infect Immun. 2010, 78: 1135-1146. 10.1128/IAI.00940-09.PubMedPubMed CentralView ArticleGoogle Scholar
- Madi A, Alnabhani Z, Leneveu C, Mijouin L, Feuilloley M, Connil N: Pseudomonas fluorescens can induce and divert the human β-defensin-2 secretion in intestinal epithelial cells to enhance its virulence. Arch Microbiol. 2013, 195: 189-195. 10.1007/s00203-012-0865-3.PubMedView ArticleGoogle Scholar
- Fu Y, Galan JE: The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton. Mol Microbiol. 1998, 27: 359-368. 10.1046/j.1365-2958.1998.00684.x.PubMedView ArticleGoogle Scholar
- Garrity-Ryan L, Kazmierczak B, Kowal R, Comolli J, Hauser A, Engel JN: The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages. Infect Immun. 2000, 68: 7100-7113. 10.1128/IAI.68.12.7100-7113.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Strauman MC, Harper JM, Harrington SM, Boll EJ, Nataro JP: Enteroaggregative Escherichia coli disrupts epithelial cell tight junctions. Infect Immun. 2010, 78: 4958-4964. 10.1128/IAI.00580-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Curcio D: Multidrug-resistant gram-negative bacterial infections: Are you ready for the challenge?. Curr Clin Pharmacol. 2013, [Epub ahead of print]Google Scholar
- Giani T, Marchese A, Coppo E, Kroumova V, Rossolini GM: VIM-1-producing Pseudomonas mosselii isolates in Italy, predating known VIM-producing index strains. Antimicrob Agents Chemother. 2012, 56: 2216-2217. 10.1128/AAC.06005-11.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.