Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Distribution of the type III effector proteins-encoding genes among nosocomial Pseudomonas aeruginosa isolates from Bulgaria

Distribution of the type III effector proteins-encoding genes among nosocomial Pseudomonas... Ann Microbiol (2010) 60:503–509 DOI 10.1007/s13213-010-0079-3 ORIGINAL PAPER Distribution of the type III effector proteins-encoding genes among nosocomial Pseudomonas aeruginosa isolates from Bulgaria Tanya Strateva & Boyka Markova & Dobrinka Ivanova & Ivan Mitov Received: 7 April 2010 /Accepted: 10 May 2010 /Published online: 12 June 2010 Springer-Verlag and the University of Milan 2010 Abstract The aim of this study was to determine the Keywords Pseudomonas aeruginosa prevalence of type III effector proteins (ExoS, ExoU, ExoT Type III effector proteins-encoding genes and ExoY)-encoding genes among clonally unrelated Polymerase chain reaction Prevalence nosocomial Pseudomonas aeruginosa strains and to analyze their distribution in respect to the infection site and antimicrobial resistance. Polymerase chain reaction-based Introduction detection of the genes was performed on 176 non-duplicate P. aeruginosa isolates from three University hospitals in Pseudomonas aeruginosa is a major cause of hospital- Sofia, previously genotyped by random amplified poly- acquired infections and the most significant pulmonary morphic DNA technique. The prevalence of the studied pathogen in cystic fibrosis patients (Pinheiro et al. 2008; genes was as follows: exoS–61.9%, exoU–32.4%, exoT– Rajan and Saiman 2002). P. aeruginosa is responsible for 100%, and exoY–85.8%. The part of P. aeruginosa strains 10–15% of nosocomial infections worldwide (Blanc et al. harboring either the exoS (54.0%) or the exoU (23.8%) gene 1998). It is not surprising that these illnesses are associated was higher (P<0.001) than that of isolates containing both with significant morbidity and mortality due to the organism’s genes (8.5%). The gene dissemination varied according to capacity to adapt easily to changes in the environment, to the infection localization. The exoU gene manifested a rapidly develop resistance to antibiotics, and to produce an higher spread (P<0.001) among multidrug-resistant (MDR) arsenal of virulence factors (Strateva 2008). than in non-MDR strains (42.6 vs 18.7%). In conclusion, the To cause such a severe disease, P. aeruginosa utilizes a P. aeruginosa type III secretion system is present in nearly large number of secreted and cell-associated virulence all studied isolates but the individual isolates from distinct factors (Van Delden and Iglewski 1998). An important infection sites differ in their effector genotypes. The ubiquity and recently recognized virulence determinant is the of type III effector proteins-encoding genes among clinical complex type III secretion system which injects effector isolates is consistent with an important role for this system in proteins into host cells (Hauser 2009). The genes encoding P. aeruginosa pathogenesis. the secretion, translocation and regulatory machinery of this system are clustered together in the 55-min region of the P. aeruginosa chromosome (http://www.pseudomonas.com). : : T. Strateva (*) B. Markova I. Mitov In contrast to the clustered genes encoding the type III Department of Medical Microbiology, transport machinery, the genes encoding the type III Medical University of Sofia, effector proteins appear to be scattered throughout the 2 Zdrave str., chromosome (Stover et al. 2000). To date, four effector 1431 Sofia, Bulgaria e-mail: dr.strateva@abv.bg proteins have been identified: ExoS (exoenzyme S), ExoU (exoenzyme U), ExoT (exoenzyme T) and ExoY (exoen- D. Ivanova zyme Y) (Engel and Balachandran 2009). ExoS and ExoT Laboratory of Clinical Microbiology, are closely related bifunctional proteins with N-terminal Second Multiprofile Active Treatment Hospital, GTPase-activating protein activity toward Rho family Sofia, Bulgaria 504 Ann Microbiol (2010) 60:503–509 proteins and C-terminal ADP ribosylase activity toward the illustra bacteria genomicPrep Mini Spin Kit (GE distinct and non-overlapping set of targets (Goehring et al. Healthcare) according to the manufacturer’s guidelines. 1999; Krall et al. 2000). ExoY is an adenylate cyclase that The DNA concentration range was about 8 μg/ml. increases intracellular levels of cAMP (Yahr et al. 1998). Intoxication with ExoS, ExoT and ExoY causes cell Polymerase chain reaction (PCR) amplification of the type III rounding and detachment and may contribute to infection effector proteins-encoding genes The genes were amplified by inhibiting or preventing bacterial uptake and phagocy- with specific primers (Alpha DNA) listed in Table 1. PCR tosis (Vallis et al. 1999). ExoU possesses phospholipase A was carried out with 2 μl template DNA, 0.25 μM of each and lysophospholipase activities (Phillips et al. 2003; primer, 0.2 mM deoxyribonucleoside triphosphates, 1× Tamura et al. 2004) that lead to rapid necrotic death in reaction buffer, 2 mM MgCl and 1.5 U Prime Taq DNA many cell types (Finck-Barbancon et al. 1997; Vallis et al. polymerase (GeNet Bio) in a total volume of 25 μl. The 1999). It is also associated with accelerated lung injury, and DNA was amplified using the following protocol: initial plays a role in the development of septic shock (Allewelt et denaturation (94°C for 5 min), followed by 25–30 cycles of al. 2000; Kurahashi et al. 1999). denaturation (94°C for 35–45 s), annealing (58–64°C, from Interestingly, the genes encoding some P. aeruginosa 45 s to 50 s), and extension (72°C, from 45 s to 1 min 30 s), type III effector proteins are found in some isolates but not with a single final extension of 7 min at 72°C. PCR products in others (Feltman et al. 2001). The distribution of these were separated in 1.5% agarose gel for 50–110 min at 130 V, genes amongst clinical isolates of P. aeruginosa remains to stained with ethidium bromide (0.5 μg/ml) and detected by be elucidated. Furthermore, the frequency of effector genes UV transillumination (wavelength 312 nm). Amplified genes in populations of isolates from different disease sites has were identified on the basis of fragment size (also shown in not been thoroughly examined. For that reason, the aim of Table 1). the present study was to determine the prevalence of the type III effector proteins-encoding genes among nosocomial DNA sequencing Selected exoS (n=10) and exoU (n=10) P. aeruginosa isolates and to analyze the values in respect to PCR products amplified from different P. aeruginosa the infection localization and antimicrobial resistance. isolates were purified by an ExoSAP-IT reagent (Amer- sham Biosciences). Sequencing reactions were performed using the sequencing primers listed in Table 1 (Strateva Materials and methods 2008), the primers for amplification and a BigDye terminator v3.1 kit (Applera) in an automated sequencer Bacterial isolates A total of 176 nosocomial isolates of (ABI 310 sequence genetic analyzer; Applied Biosystems). P. aeruginosa was used in the present study. They were The nucleotide and deduced amino acid sequences were cultured during 2001–2009 from hospitalized patients (n= analyzed with software available from the National Center 176) of different types of ward in three University for Biotechnology Information (http://www.ncbi.nlm.nih. hospitalsinSofia andwereobtainedasfollows:from gov). The GenBank accession numbers of the published urine (n=67), tracheal aspirates (n=26), bronchial lavage sequences are: AY029250 (P. aeruginosa exoS gene) and (n=12), sputum (n=12), pleural fluid (n=2), surgical U97065 (P. aeruginosa ExoU operon). wounds or abscesses (n=29), blood (n=8), nose (n=9), throat (n=8), and bile (n=3). Bacterial identification was Random amplified polymorphic DNA (RAPD) analysis - performed using a BBL Enteric/Nonfermenter ID system RAPD was performed with Ready-To-Go RAPD Analysis (Becton Dickinson). Beads (GE Healthcare) according to the manufacturer’s guidelines. The amplifications were carried out with 2 μl Definition of multidrug-resistant (MDR) isolates MDR DNA and 1 μl primer (RAPD-4 5′-AAGAGCCCGT-3′)ina P. aeruginosa isolates were defined as resistant to one or total volume of 25 μl. The following protocol was applied: more representatives of at least three antibiotic classes with 1 cycle of 5 min at 95°C, followed by 45 cycles of antipseudomonal activity (β-lactams, aminoglycosides and denaturation for 1 min at 95°C, annealing of primers for fluoroquinolones) (Wang et al. 2006; Pagani et al. 2005; 1 min at 36°C ,and elongation for 2 min at 72°C. The Kirikae et al. 2008), using the conventional serial agar amplifications products were compared by electrophoresis dilution method. The minimal inhibitory concentrations of 10-μl samples in 2% agarose gel stained with ethidium were interpreted according to the Clinical and Laboratory bromide and photographed under UV light. The bends Standards Institute (CLSI) 2007 guide-line (CLSI 2007). generated were analyzed using Dice coefficient (similarity coefficient, S ) for every pair of isolates. Isolates with AB DNA isolation Bacterial DNA isolation was performed S value of >70% are considered to be closely related. AB from a single colony on Brucella sheep blood agar using Two E. coli strain (BL21 (DE3) and C1a) DNAs were used Ann Microbiol (2010) 60:503–509 505 Table 1 Primers used for amplification and sequencing of the type III effector proteins-encoding genes Primers Target Sequence (5′–3′) Product size (bp) Position on P. aeruginosa Annealing Source gene PAO1 chromosome (locus) temperature (ºC) Pairs for PCR exoS-F exoS CTT GAA GGG ACT CGA CAA GG 504 PA3841 60 Lanotte et al. 2004 exoS-R TTC AGG TCC GCG TAG TGA AT exoT-F exoT CAA TCA TCT CAG CAG AAC CC 1159 PA0044 58 Finnan et al. 2004 exoT-R TGT CGT AGA GGA TCT CCT G exoY-F exoY TAT CGA CGG TCA TCG TCA GGT 1035 PA2191 64 Finnan et al. 2004 exoY-R TTG ATG CAC TCG ACC AGC AAG exoU-F exoU GGG AAT ACT TTC CGG GAA GTT 428 PS14 within P. aeruginosa 60 Allewelt et al. 2000 PA14 pathogenicity island PAPI-2 exoU-R CGA TCT CGC TGC TAA TGT GTT Sequencing primers exoS-F-seq exoS ATG CAT ATT CAA TCG CTT CA Strateva 2008 exoS-R-seq exoS CGA CCG GTC AGG CCA GAT Strateva 2008 exoU-R-seq1 exoU TCA TGT GAA CTC CTT ATT Strateva 2008 exoU-R-seq2 exoU CGA GAG AAG CGA AGG TAT GA Strateva 2008 PCR Polymerase chain reaction, F forward, R reverse exoS exoenzyme S-encoding gene, exoT exoenzyme T-encoding gene, exoY exoenzyme Y-encoding gene, exoU exoenzyme U-encoding gene as controls to assay the ability of the RAPD beads to amplify the RAPD analysis confirmed the significant diversity of the DNA and identify polymorphisms. The control DNAs were strains included in the present study. genotyped by both RAPD-4 and RAPD-2 (5′-GTTT CGCTCC-3′)primers. Overall prevalence of the type III effector proteins-encoding genes Statistical analysis The distribution of virulence genes with respect to isolate origin was compared using Student’s t The frequencies of occurrence of these genes detected by test. A P value below 0.05 was considered to be statistically PCR in all studied strains (n=176) were as follows: exoS– significant. 61.9%, exoU–32.4%, exoT–100%, and exoY–85.8%. The sequences of the exoS and exoU genes amplified from different isolates were identical for all examined isolates and 100% identical to the known sequences (AY029250 Results and discussion and U97065, respectively). The prevalence of three genes (exoS, exoU and exoY) was similar to that ascertained by Evaluation of clonal relatedness of the studied Feltman et al. (2001), respectively 72, 28 and 89%, among P. aeruginosa isolates by RAPD typing clinical P. aeruginosa isolated in USA during 1999–2000. The absolute presence of exoT was not surprising. Several A total of 176 P. aeruginosa nosocomial strains isolated recent studies reported a very high prevalence of this gene during an 8-year period were analyzed by RAPD typing. among clinical and environmental isolates of P. aeruginosa RAPD fingerprints demonstrated that most strains were of (Feltman et al. 2001; Lomholt et al. 2001; Winstanley et al. distinct genotype and determined the presence of 106 RAPD 2005). patterns. A large number of bands (with size from 130 to The majority of the 176 strains included in our study 2,200 bp) were generated by the RAPD-4 primer. Eleven (47.2%) exhibited a combination of exoS, exoT and exoY; strains revealed unique RAPD profiles. A few strains were 21% possessed a combination of exoU, exoT and exoY closely related (S >70%)-9 nosocomial P. aeruginosa (Table 2). The part of P. aeruginosa strains containing either AB isolates from 4 different clinics of the University hospital the exoS (54.0%) or the exoU gene (28.3%) was significantly No 1 (with S value of 82%-Fig. 1), 14 isolates from the higher (P<0.001) than the part of isolates harboring both AB Neonatology clinic (86%) and 8 isolates from the Urology genes exoS+exoU (8.5%). Previously, Feltman et al. (2001) clinic (82.6%) of the University hospital No 2. In summary, reported that of 115 P. aeruginosa clinical and environmental 506 Ann Microbiol (2010) 60:503–509 Fig. 1 Randomly amplified K1 1 2 3 4 5 6 7 8 9 M 10 11 12 13 14 15 16 17 K2 polymorphic DNA (RAPD) of P. aeruginosa isolates generated with RAPD-4 primer. Lanes: K1 RAPD profile of the reference strain E. coli BL21 (DE3) generated with RAPD-2 primer, K2 RAPD profile of the reference strain E. coli C1a generated with RAPD-2 primer, M standard size marker (100-bp ladder), 1–17 nosocomial P. aeruginosa isolates. Lanes 2–8, 10 and 11 show the RAPD profiles of nine closely related strains (S value of 82%) from AB four different clinics isolates, 82 contained the exoS but not the exoU gene, 31 (3) The exoY gene was disseminated among all studied contained the exoU but not the exoS gene and a single isolates from blood, which was higher than the exoY- contained both genes. Two recent studies demonstrated a distribution in P. aeruginosa isolates from urine very low frequency of occurrence of P. aeruginosa blood- (85.1%, P<0.001), LRTIs (82.7%, P<0.01) and stream isolates harboring both genes, respectively 2.2 and wound (82.8%, P<0.02). 1.6% (Berthelot et al. 2003; Garey et al. 2008). Winstanley et al. (2005) established a similar find among P. aeruginosa The prevalence of the exoS among our LRTIs-isolates isolates associated with ulcerative keratitis. In a recent (53.8%) was lower than that in LRTIs P. aeruginosa multicenter study conducted by Pirnay et al. (2009), 72.6% isolates from the studies mentioned above (Lanotte et al. of all P. aeruginosa isolates harbored the exoS gene, 23.1% 2004, 80%; Feltman et al. 2001, 75%). ExoS is responsible contained the exoU gene and with the exception of three for direct tissue destruction in lung infection and may be strains, the carriage of exoU and exoS was mutually important for bacterial dissemination (Nicas et al. 1985a; b). exclusive. In contrast, Finnan et al. (2004) found 75% In this study, the distribution of the exoU gene was dissemination of both genes in clinical and environmental very heterogeneous. The gene was found predominantly in P. aeruginosa isolates. P. aeruginosa isolates from LRTIs and wounds. Its prevalence in our wound isolates (51.7%) was similar to that Prevalence of the type III effector proteins-encoding genes ascertained by Feltman et al. (2001) in the USA (40.0%). In with respect to the infection localization contrast, Lomholt et al. (2001) reported an absence of the The distribution of virulence genes encoding the type III Table 2 Distribution of the exo gene patterns among nosocomial effector proteins varied in respect to the infection localization P. aeruginosa isolates (n=176) (Table 3). Gene pattern Number (%) of isolates (1) The presence of exoS was the highest among P. aeruginosa isolates from blood (87.5%) and − − + − exoS /exoU /exoT /exoY 4 (2.3) + − + − significantly different only with those obtained from exoS /exoU /exoT /exoY 12 (6.8) in-patients with lower respiratory tract infections − + + − exoS /exoU /exoT /exoY 5 (2.8) (LRTIs) (53.8%)-P<0.02. − − + + exoS /exoU /exoT /exoY 20 (11.4) (2) The exoU frequencies were the most expressive in the + − + + exoS /exoU /exoT /exoY 83 (47.2) wound (51.7%) and LRTIs P. aeruginosa isolates − + + + exoS /exoU /exoT /exoY 37 (21.0) (40.4%). They were significantly higher than those of + + + − exoS /exoU /exoT /exoY 3 (1.7) the isolates from urine (19.4%), P<0.01 and P<0.02, + + + + exoS /exoU /exoT /exoY 12 (6.8) respectively, and blood (12.5%), P<0.01 and P<0.05, respectively. exo Genes encoding type III effector proteins, + presence, − absence Ann Microbiol (2010) 60:503–509 507 Table 3 Prevalence (as percentage) of the type III effector proteins-encoding genes in nosocomial P. aeruginosa isolates, in respect to the infection site Gene Isolate origin Urine (n=67) LRTIs (n=52) URTIs (n=17) Wounds (n=29) Blood (n=8) Total (n=176) exoS 62.7 53.8 70.6 62.1 87.5 61.9 exoU 19.4 40.4 35.3 51.7 12.5 32.4 exoT 100.0 100.0 100.0 100.0 100.0 100.0 exoY 85.1 82.7 94.1 82.8 100.0 85.8 LRTIs Lower respiratory tract infections, URTIs upper respiratory tract infections, exoS exoenzyme S-encoding gene, exoU exoenzyme U-encoding gene, exoT exoenzyme T-encoding gene, exoY exoenzyme Y-encoding gene Frequency of the type III effector gene in all studied isolates of P. aeruginosa, including three bile isolates exoU in a small series of 11 wound isolates of P. aeruginosa to harbor the exoU gene and to display high swimming (Lomholt et al. 2001). Our urine isolates showed a low motility and adhesiveness. percentage of exoU carriers (19.4%), while Lomholt et al. Recently, it was reported the worldwide spread and did not find urine isolates harboring the exoU gene. persistence of MDR clone comprising P. aeruginosa O12 + − It is known that 90% of ExoU-producing P. aeruginosa isolates which exhibited the exoS /exoU genotype (Pirnay strains are associated with severe infections (Hauser et al. et al. 2009). Moreover, most MDR clinical isolates of 2002). Of the type III secretion proteins, ExoU is the P. aeruginosa reveal either O11 or O12 serotype (Sekiguchi most cytotoxic. Its secretion is a marker for highly et al. 2007; Pirnay et al. 2009). Evaluation of the spread of virulent P. aeruginosa isolates obtained from patients with these serotypes among Bulgarian nosocomial MDR hospital-acquired pneumonia (Schulert et al. 2003). P. aeruginosa strains, as well as their corresponding effector genotypes, should be objectives of our future investigations. Prevalence of the type III effector proteins-encoding genes Whether MDR P. aeruginosa strains necessarily express with respect to the antimicrobial resistance a more virulent phenotype continues to remain a contro- versial issue (Di Martino et al. 2002). Of the 176 nosocomial P. aeruginosa isolates, 101 (57.4%) In conclusion, our results suggest that type III secretion were MDR. One of the genes, exoU manifested a genes are found in nearly all investigated P. aeruginosa but significantly higher spread (P<0.001) among MDR than that individual isolates from distinct infection sites differ in in non-MDR strains of P. aeruginosa (42.6 vs 18.7%) their effector genotypes. Our data confirm that the exoU (Fig. 2). Twelve isolates contained the four studied genes (Table 2), moreover 8 of them were MDR. 100.00% In a recent accomplished study, Garey et al. (2008) determined the prevalence of exoU and exoS from blood- 80.00% stream isolates of hospitalized patients with P. aeruginosa bacteremia and ascertained that the isolates containing the 60.00% exoU gene were significantly more resistant (P<0.05) to different classes of antimicrobials: β-lactams (piperacillin/ 40.00% tazobactam, ceftazidime, cefepime, carbapenems), fluoro- quinolones, and aminoglycosides (gentamicin). Other 20.00% research suggested that the multidrug resistance in ocular P. aeruginosa was more commonly associated with strains 0.00% having cytotoxicity and exoU gene, and belonging to exoS exoU exoT exoY serotype E (Zhu et al. 2006). Recently, Zaborina et al. MDR (n=101) non-MDR (n=75) (2006) screened consecutively isolated MDR P. aerugi- nosa clinical strains for their ability to disrupt the integrity Fig. 2 Prevalence (as percentages) of the type III effector proteins- of human cultured intestinal epithelial cells (Caco-2) and encoding genes among MDR and non-MDR P. aeruginosa isolates correlated these findings to related virulence phenotypes from non-cystic fibrosis patients. MDR multidrug-resisrant, exoS such as adhesiveness, motility, biofilm formation and exoenzyme S-encoding gene, exoU exoenzyme U-encoding gene, cytotoxicity. These strains were characterized and found exoT exoenzyme T-encoding gene, exoY exoenzyme Y-encoding gene 508 Ann Microbiol (2010) 60:503–509 and exoS genes are almost mutually exclusive. Nosocomial Goehring UM, Schmidt G, Pedersen KJ, Aktories K, Barbieri JT (1999) The N-terminal domain of Pseudomonas aeruginosa isolates of P. aeruginosa harboring the exoU gene were exoenzyme S is a GTPase-activating protein for Rho GTPases. more likely to be resistant to multiple antibiotics. The J Biol Chem 274:36369–36372 ubiquity of type III effector proteins-encoding genes among Hauser A (2009) The type III secretion system of Pseudomonas clinical isolates is consistent with an important role for this aeruginosa: infection by injection. Nat Rev Microbiol 7:654–665 Hauser AR, Cobb E, Bodi M, Mariscal D, Valles J, Engel JN, system in human disease. Future studies to determine the Rello J (2002) Type III protein secretion is associated with prevalence of clinical isolates capable of secreting specific poor clinical outcomes in patients with ventilator-associated effector proteins and whether secretion correlates with pneumonia caused by Pseudomonas aeruginosa.CritCare Med 30:521–528 infection prognosis are necessary for evaluation of the type Kirikae T, Mizuguchi Y, Arakawa Y (2008) Investigation of isolation III secretion system in P. aeruginosa pathogenesis. rates of Pseudomonas aeruginosa with and without multidrug resistance in medical facilities and clinical laboratories in Japan. J Acknowledgement This work was supported by a grant from the Antimicrob Chemother 61:612–615 Medical University of Sofia (Council of Medical Science, project no. Krall R, Schmidt G, Aktories K, Barbieri JT (2000) Pseudomonas 4/2009, grant no. 20/2009). aeruginosa ExoT is a Rho GTPase-activating protein. Infect The authors declare that they have no conflict of interest. Immun 68:6066–6068 Kurahashi K, Kajikawa O, Sawa T, Ohara M, Gropper MA, Frank DW, Martin TR, Wiener-Kronish JP (1999) Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J Clin References Invest 104:743–750 Lanotte P, Watt S, Mereghetti L, Dartiguelongue N, Rastegar-Lari A, Allewelt M, Coleman FT, Grout M, Priebe GP, Pier GB (2000) Goudeau A, Quentin R (2004) Genetic feature of Pseudomonas Acquisition of expression of the Pseudomonas aeruginosa ExoU aeruginosa isolates from cystic fibrosis patients compared with cytotoxin leads to increased bacterial virulence in a murine model those of isolates from other origins. J Med Microbiol 53:73–81 of acute pneumonia and systemic spread. Infect Immun 68:3998– Lomholt JA, Poulsen K, Kilian M (2001) Epidemic population 4004 structure of Pseudomonas aeruginosa: evidence for a clone that Berthelot P, Attree I, Plésiat P, Chabert J, de Bentzmann S, Pozzetto B, is pathogenic to the eye and that has a distinct combination of Grattard F, the Groupe d’Etudes des Septicémies à Pseudomonas virulence factors. Infect Immun 69:6284–6295 aeruginosa (2003) Genotypic and phenotypic analysis of type III Nicas TI, Bradley J, Lochner JE, Iglewski BH (1985a) The role of secretion system in a cohort of Pseudomonas aeruginosa exoenzyme S in infections with Pseudomonas aeruginosa.J bacteremia isolates: evidence for a possible association between Infect Dis 152:716–721 O serotypes and exo genes. J Infect Dis 188:512–518 Nicas TI, Frank DW, Stenzel P, Lile JD, Iglewski BH (1985b) Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. Blanc DS, Petignat C, Janin B, Bille J, Francioli P (1998) Frequency Eur J Clin Microbiol 4:175–179 and molecular diversity of Pseudomonas aeruginosa upon Pagani L, Colinon C, Migliavacca R, Labonia M, Docquier JD, admission and during hospitalization: a prospective epidemiologic Nucleo E, Spalla M, Li Bergoli M, Rossolini GM (2005) study. Clin Microbiol Infect 4:242–247 Nosocomial outbreak caused by multidrug-resistant Pseudomonas CLSI (2007) Performance standards for antimicrobial susceptibility testing, 17th informational supplement, M100-S17. Clinical and aeruginosa producing IMP-13 metallo-β-lactamase. J Clin Laboratory Standards Institute, Wayne Microbiol 43:3824–3828 Di Martino P, Gagniere H, Berry H, Bret L (2002) Antibiotic Phillips RM, Six DA, Dennis EA, Ghosh P (2003) In vivo resistance and virulence properties of Pseudomonas aeruginosa phospholipase activity of the Pseudomonas aeruginosa cytotoxin strains from mechanically ventilated patients with pneumonia in ExoU and protection of mammalian cells with phospholipase A intensive care units: comparison with imipenem-resistant inhibitors. J Biol Chem 278:41326–41332 extra-respiratory tract isolates from uninfected patients. Pinheiro MRS, Lacerda HR, Melo RGL, Maciel MA (2008) Microbes Infect 4:613–620 Pseudomonas aeruginosa infections: factors relating to mortality Engel J, Balachandran P (2009) Role of Pseudomonas aeruginosa with emphasis on resistance pattern and antimicrobial treatment. type III effectors in disease. Curr Opin Microbiol 12:61–66 Braz J Infect Dis 12:509–515 Feltman H, Schulert G, Khan S, Jain M, Peterson L, Hauser AR Pirnay J-P, Bilocq F, Pot B, Cornelis P, Zizi M, Van Eldere J, (2001) Prevalence of type III secretion genes in clinical and Deschaght P, Vaneechoutte M, Jennes S, Pitt T, De Voss D (2009) environmental isolates of Pseudomonas aeruginosa. Microbiology Pseudomonas aeruginosa population structure revisited. PLoS 147:2659–2669 ONE 4:e7740. doi:10.1371/journal.pone.0007740 Finck-Barbancon V, Goranson J, Zhu L, Sawa T, Wiener-Kronish JP, Rajan S, Saiman L (2002) Pulmonary infections in patients with cystic Fleiszig SM, Wu C, Mende-Mueller L, Frank DW (1997) ExoU fibrosis. Semin Respir Infect 17:47–56 expression by Pseudomonas aeruginosa correlates with acute Schulert GS, Feltman H, Rabin SD, Martin CG, Battle SE, Rello J, cytotoxicity and epithelial injury. Mol Microbiol 25:547–557 Hauser AR (2003) Secretion of the toxin ExoU is a marker for Finnan S, Morrissey JP, O’Gara F, Boyd EF (2004) Genome highly virulent Pseudomonas aeruginosa isolates obtained from diversity of Pseudomonas aeruginosa isolates from cystic patients with hospital-acquired pneumonia. J Infect Dis fibrosis patients and the hospital environment. J Clin Microbiol 188:1695–1706 42:5783–5792 Sekiguchi J, Asagi T, Miyoshi-Akiyama T, Kasai A, Mizuguchi Y, Araake M, Fujino T, Kikuchi H, Sasaki S, Watari H, Kojima T, Garey KW, Vo QP, Larocco MT, Gentry LO, Tam VH (2008) Miki H, Kanemitsu K, Kunishima H, Kikuchi Y, Kaku M, Prevalence of type III secretion protein exoenzymes and Yoshikura H, Kuratsuji T, Kirikae T (2007) Outbreaks of antimicrobial susceptibility patterns from bloodstream isolates multidrug-resistant Pseudomonas aeruginosa in community of patients with Pseudomonas aeruginosa bacteremia. J hospitals in Japan. J Clin Microbiol 45:979–989 Chemother 20:714–720 Ann Microbiol (2010) 60:503–509 509 Stover CK, Pham XQ, Erwin AL et al (2000) Complete genome hospitalised patients: clinical features, risk-factors and outcomes. sequence of Pseudomonas aeruginosa PAO1, an opportunistic Clin Microbiol Infect 12:63–68 pathogen. Nature 406:959–964 Winstanley C, Kaye SB, Neal TJ, Chilton HJ, Miksch S, Hart CA, Strateva T (2008) Microbiological and molecular-genetic investiga- Microbiology Ophthalmic Group (2005) Genotypic and phenotypic tions on resistance mechanisms and virulence factors in clinical characteristics of Pseudomonas aeruginosa isolates associated with strains of Pseudomonas aeruginosa. PhD dissertation, Medical ulcerative keratitis. J Med Microbiol 54:519–526 University of Sofia, Bulgaria Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW (1998) ExoY, Tamura M, Ajayi T, Allmond LR, Moriyama K, Wiener-Kronish JP, an adenylate cyclase secreted by the Pseudomonas aeruginosa type Sawa T (2004) Lysophospholipase A activity of Pseudomonas III system. Proc Natl Acad Sci USA 95:13899–13904 aeruginosa type III secretory toxin ExoU. Biochem Biophys Res Zaborina O, Kohler JE, Wang Y, Bethel C, Shevchenko O, Wu L, Commun 316:323–331 Turner JR, Alverdy JC (2006) Identification of multi-drug Vallis AJ, Fink-Barbacon V, Yahr TL, Frank DW (1999) Biological resistant Pseudomonas aeruginosa clinical isolates that are effects of Pseudomonas aeruginosa type III-secreted proteins on highly disruptive to the intestinal epithelial barrier. Ann Clin CHO cells. Infect Immun 67:2040–2044 Microbiol Antimicrob 5:14 Van Delden C, Iglewski BH (1998) Cell-to-cell signaling and Zhu H, Conibear TCR, Bandara R, Aliwarga Y, Stapleton F, Willcox Pseudomonas aeruginosa infections. Emerg Infect Dis 4:551– MDP (2006) Type III secretion system-associated toxins, 560 proteases, serotypes, and antibiotic resistance of Pseudomonas Wang CY, Jerng JS, Cheng KY, Lee LN, Yu CJ, Hsueh PR, Yang PC aeruginosa isolates associated with keratitis. Curr Eye Res (2006) Pandrug-resistant Pseudomonas aeruginosa among 31:297–306 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Distribution of the type III effector proteins-encoding genes among nosocomial Pseudomonas aeruginosa isolates from Bulgaria

Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/distribution-of-the-type-iii-effector-proteins-encoding-genes-among-NgvrSyhwCo

References (39)

Publisher
Springer Journals
Copyright
Copyright © 2010 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-010-0079-3
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2010) 60:503–509 DOI 10.1007/s13213-010-0079-3 ORIGINAL PAPER Distribution of the type III effector proteins-encoding genes among nosocomial Pseudomonas aeruginosa isolates from Bulgaria Tanya Strateva & Boyka Markova & Dobrinka Ivanova & Ivan Mitov Received: 7 April 2010 /Accepted: 10 May 2010 /Published online: 12 June 2010 Springer-Verlag and the University of Milan 2010 Abstract The aim of this study was to determine the Keywords Pseudomonas aeruginosa prevalence of type III effector proteins (ExoS, ExoU, ExoT Type III effector proteins-encoding genes and ExoY)-encoding genes among clonally unrelated Polymerase chain reaction Prevalence nosocomial Pseudomonas aeruginosa strains and to analyze their distribution in respect to the infection site and antimicrobial resistance. Polymerase chain reaction-based Introduction detection of the genes was performed on 176 non-duplicate P. aeruginosa isolates from three University hospitals in Pseudomonas aeruginosa is a major cause of hospital- Sofia, previously genotyped by random amplified poly- acquired infections and the most significant pulmonary morphic DNA technique. The prevalence of the studied pathogen in cystic fibrosis patients (Pinheiro et al. 2008; genes was as follows: exoS–61.9%, exoU–32.4%, exoT– Rajan and Saiman 2002). P. aeruginosa is responsible for 100%, and exoY–85.8%. The part of P. aeruginosa strains 10–15% of nosocomial infections worldwide (Blanc et al. harboring either the exoS (54.0%) or the exoU (23.8%) gene 1998). It is not surprising that these illnesses are associated was higher (P<0.001) than that of isolates containing both with significant morbidity and mortality due to the organism’s genes (8.5%). The gene dissemination varied according to capacity to adapt easily to changes in the environment, to the infection localization. The exoU gene manifested a rapidly develop resistance to antibiotics, and to produce an higher spread (P<0.001) among multidrug-resistant (MDR) arsenal of virulence factors (Strateva 2008). than in non-MDR strains (42.6 vs 18.7%). In conclusion, the To cause such a severe disease, P. aeruginosa utilizes a P. aeruginosa type III secretion system is present in nearly large number of secreted and cell-associated virulence all studied isolates but the individual isolates from distinct factors (Van Delden and Iglewski 1998). An important infection sites differ in their effector genotypes. The ubiquity and recently recognized virulence determinant is the of type III effector proteins-encoding genes among clinical complex type III secretion system which injects effector isolates is consistent with an important role for this system in proteins into host cells (Hauser 2009). The genes encoding P. aeruginosa pathogenesis. the secretion, translocation and regulatory machinery of this system are clustered together in the 55-min region of the P. aeruginosa chromosome (http://www.pseudomonas.com). : : T. Strateva (*) B. Markova I. Mitov In contrast to the clustered genes encoding the type III Department of Medical Microbiology, transport machinery, the genes encoding the type III Medical University of Sofia, effector proteins appear to be scattered throughout the 2 Zdrave str., chromosome (Stover et al. 2000). To date, four effector 1431 Sofia, Bulgaria e-mail: dr.strateva@abv.bg proteins have been identified: ExoS (exoenzyme S), ExoU (exoenzyme U), ExoT (exoenzyme T) and ExoY (exoen- D. Ivanova zyme Y) (Engel and Balachandran 2009). ExoS and ExoT Laboratory of Clinical Microbiology, are closely related bifunctional proteins with N-terminal Second Multiprofile Active Treatment Hospital, GTPase-activating protein activity toward Rho family Sofia, Bulgaria 504 Ann Microbiol (2010) 60:503–509 proteins and C-terminal ADP ribosylase activity toward the illustra bacteria genomicPrep Mini Spin Kit (GE distinct and non-overlapping set of targets (Goehring et al. Healthcare) according to the manufacturer’s guidelines. 1999; Krall et al. 2000). ExoY is an adenylate cyclase that The DNA concentration range was about 8 μg/ml. increases intracellular levels of cAMP (Yahr et al. 1998). Intoxication with ExoS, ExoT and ExoY causes cell Polymerase chain reaction (PCR) amplification of the type III rounding and detachment and may contribute to infection effector proteins-encoding genes The genes were amplified by inhibiting or preventing bacterial uptake and phagocy- with specific primers (Alpha DNA) listed in Table 1. PCR tosis (Vallis et al. 1999). ExoU possesses phospholipase A was carried out with 2 μl template DNA, 0.25 μM of each and lysophospholipase activities (Phillips et al. 2003; primer, 0.2 mM deoxyribonucleoside triphosphates, 1× Tamura et al. 2004) that lead to rapid necrotic death in reaction buffer, 2 mM MgCl and 1.5 U Prime Taq DNA many cell types (Finck-Barbancon et al. 1997; Vallis et al. polymerase (GeNet Bio) in a total volume of 25 μl. The 1999). It is also associated with accelerated lung injury, and DNA was amplified using the following protocol: initial plays a role in the development of septic shock (Allewelt et denaturation (94°C for 5 min), followed by 25–30 cycles of al. 2000; Kurahashi et al. 1999). denaturation (94°C for 35–45 s), annealing (58–64°C, from Interestingly, the genes encoding some P. aeruginosa 45 s to 50 s), and extension (72°C, from 45 s to 1 min 30 s), type III effector proteins are found in some isolates but not with a single final extension of 7 min at 72°C. PCR products in others (Feltman et al. 2001). The distribution of these were separated in 1.5% agarose gel for 50–110 min at 130 V, genes amongst clinical isolates of P. aeruginosa remains to stained with ethidium bromide (0.5 μg/ml) and detected by be elucidated. Furthermore, the frequency of effector genes UV transillumination (wavelength 312 nm). Amplified genes in populations of isolates from different disease sites has were identified on the basis of fragment size (also shown in not been thoroughly examined. For that reason, the aim of Table 1). the present study was to determine the prevalence of the type III effector proteins-encoding genes among nosocomial DNA sequencing Selected exoS (n=10) and exoU (n=10) P. aeruginosa isolates and to analyze the values in respect to PCR products amplified from different P. aeruginosa the infection localization and antimicrobial resistance. isolates were purified by an ExoSAP-IT reagent (Amer- sham Biosciences). Sequencing reactions were performed using the sequencing primers listed in Table 1 (Strateva Materials and methods 2008), the primers for amplification and a BigDye terminator v3.1 kit (Applera) in an automated sequencer Bacterial isolates A total of 176 nosocomial isolates of (ABI 310 sequence genetic analyzer; Applied Biosystems). P. aeruginosa was used in the present study. They were The nucleotide and deduced amino acid sequences were cultured during 2001–2009 from hospitalized patients (n= analyzed with software available from the National Center 176) of different types of ward in three University for Biotechnology Information (http://www.ncbi.nlm.nih. hospitalsinSofia andwereobtainedasfollows:from gov). The GenBank accession numbers of the published urine (n=67), tracheal aspirates (n=26), bronchial lavage sequences are: AY029250 (P. aeruginosa exoS gene) and (n=12), sputum (n=12), pleural fluid (n=2), surgical U97065 (P. aeruginosa ExoU operon). wounds or abscesses (n=29), blood (n=8), nose (n=9), throat (n=8), and bile (n=3). Bacterial identification was Random amplified polymorphic DNA (RAPD) analysis - performed using a BBL Enteric/Nonfermenter ID system RAPD was performed with Ready-To-Go RAPD Analysis (Becton Dickinson). Beads (GE Healthcare) according to the manufacturer’s guidelines. The amplifications were carried out with 2 μl Definition of multidrug-resistant (MDR) isolates MDR DNA and 1 μl primer (RAPD-4 5′-AAGAGCCCGT-3′)ina P. aeruginosa isolates were defined as resistant to one or total volume of 25 μl. The following protocol was applied: more representatives of at least three antibiotic classes with 1 cycle of 5 min at 95°C, followed by 45 cycles of antipseudomonal activity (β-lactams, aminoglycosides and denaturation for 1 min at 95°C, annealing of primers for fluoroquinolones) (Wang et al. 2006; Pagani et al. 2005; 1 min at 36°C ,and elongation for 2 min at 72°C. The Kirikae et al. 2008), using the conventional serial agar amplifications products were compared by electrophoresis dilution method. The minimal inhibitory concentrations of 10-μl samples in 2% agarose gel stained with ethidium were interpreted according to the Clinical and Laboratory bromide and photographed under UV light. The bends Standards Institute (CLSI) 2007 guide-line (CLSI 2007). generated were analyzed using Dice coefficient (similarity coefficient, S ) for every pair of isolates. Isolates with AB DNA isolation Bacterial DNA isolation was performed S value of >70% are considered to be closely related. AB from a single colony on Brucella sheep blood agar using Two E. coli strain (BL21 (DE3) and C1a) DNAs were used Ann Microbiol (2010) 60:503–509 505 Table 1 Primers used for amplification and sequencing of the type III effector proteins-encoding genes Primers Target Sequence (5′–3′) Product size (bp) Position on P. aeruginosa Annealing Source gene PAO1 chromosome (locus) temperature (ºC) Pairs for PCR exoS-F exoS CTT GAA GGG ACT CGA CAA GG 504 PA3841 60 Lanotte et al. 2004 exoS-R TTC AGG TCC GCG TAG TGA AT exoT-F exoT CAA TCA TCT CAG CAG AAC CC 1159 PA0044 58 Finnan et al. 2004 exoT-R TGT CGT AGA GGA TCT CCT G exoY-F exoY TAT CGA CGG TCA TCG TCA GGT 1035 PA2191 64 Finnan et al. 2004 exoY-R TTG ATG CAC TCG ACC AGC AAG exoU-F exoU GGG AAT ACT TTC CGG GAA GTT 428 PS14 within P. aeruginosa 60 Allewelt et al. 2000 PA14 pathogenicity island PAPI-2 exoU-R CGA TCT CGC TGC TAA TGT GTT Sequencing primers exoS-F-seq exoS ATG CAT ATT CAA TCG CTT CA Strateva 2008 exoS-R-seq exoS CGA CCG GTC AGG CCA GAT Strateva 2008 exoU-R-seq1 exoU TCA TGT GAA CTC CTT ATT Strateva 2008 exoU-R-seq2 exoU CGA GAG AAG CGA AGG TAT GA Strateva 2008 PCR Polymerase chain reaction, F forward, R reverse exoS exoenzyme S-encoding gene, exoT exoenzyme T-encoding gene, exoY exoenzyme Y-encoding gene, exoU exoenzyme U-encoding gene as controls to assay the ability of the RAPD beads to amplify the RAPD analysis confirmed the significant diversity of the DNA and identify polymorphisms. The control DNAs were strains included in the present study. genotyped by both RAPD-4 and RAPD-2 (5′-GTTT CGCTCC-3′)primers. Overall prevalence of the type III effector proteins-encoding genes Statistical analysis The distribution of virulence genes with respect to isolate origin was compared using Student’s t The frequencies of occurrence of these genes detected by test. A P value below 0.05 was considered to be statistically PCR in all studied strains (n=176) were as follows: exoS– significant. 61.9%, exoU–32.4%, exoT–100%, and exoY–85.8%. The sequences of the exoS and exoU genes amplified from different isolates were identical for all examined isolates and 100% identical to the known sequences (AY029250 Results and discussion and U97065, respectively). The prevalence of three genes (exoS, exoU and exoY) was similar to that ascertained by Evaluation of clonal relatedness of the studied Feltman et al. (2001), respectively 72, 28 and 89%, among P. aeruginosa isolates by RAPD typing clinical P. aeruginosa isolated in USA during 1999–2000. The absolute presence of exoT was not surprising. Several A total of 176 P. aeruginosa nosocomial strains isolated recent studies reported a very high prevalence of this gene during an 8-year period were analyzed by RAPD typing. among clinical and environmental isolates of P. aeruginosa RAPD fingerprints demonstrated that most strains were of (Feltman et al. 2001; Lomholt et al. 2001; Winstanley et al. distinct genotype and determined the presence of 106 RAPD 2005). patterns. A large number of bands (with size from 130 to The majority of the 176 strains included in our study 2,200 bp) were generated by the RAPD-4 primer. Eleven (47.2%) exhibited a combination of exoS, exoT and exoY; strains revealed unique RAPD profiles. A few strains were 21% possessed a combination of exoU, exoT and exoY closely related (S >70%)-9 nosocomial P. aeruginosa (Table 2). The part of P. aeruginosa strains containing either AB isolates from 4 different clinics of the University hospital the exoS (54.0%) or the exoU gene (28.3%) was significantly No 1 (with S value of 82%-Fig. 1), 14 isolates from the higher (P<0.001) than the part of isolates harboring both AB Neonatology clinic (86%) and 8 isolates from the Urology genes exoS+exoU (8.5%). Previously, Feltman et al. (2001) clinic (82.6%) of the University hospital No 2. In summary, reported that of 115 P. aeruginosa clinical and environmental 506 Ann Microbiol (2010) 60:503–509 Fig. 1 Randomly amplified K1 1 2 3 4 5 6 7 8 9 M 10 11 12 13 14 15 16 17 K2 polymorphic DNA (RAPD) of P. aeruginosa isolates generated with RAPD-4 primer. Lanes: K1 RAPD profile of the reference strain E. coli BL21 (DE3) generated with RAPD-2 primer, K2 RAPD profile of the reference strain E. coli C1a generated with RAPD-2 primer, M standard size marker (100-bp ladder), 1–17 nosocomial P. aeruginosa isolates. Lanes 2–8, 10 and 11 show the RAPD profiles of nine closely related strains (S value of 82%) from AB four different clinics isolates, 82 contained the exoS but not the exoU gene, 31 (3) The exoY gene was disseminated among all studied contained the exoU but not the exoS gene and a single isolates from blood, which was higher than the exoY- contained both genes. Two recent studies demonstrated a distribution in P. aeruginosa isolates from urine very low frequency of occurrence of P. aeruginosa blood- (85.1%, P<0.001), LRTIs (82.7%, P<0.01) and stream isolates harboring both genes, respectively 2.2 and wound (82.8%, P<0.02). 1.6% (Berthelot et al. 2003; Garey et al. 2008). Winstanley et al. (2005) established a similar find among P. aeruginosa The prevalence of the exoS among our LRTIs-isolates isolates associated with ulcerative keratitis. In a recent (53.8%) was lower than that in LRTIs P. aeruginosa multicenter study conducted by Pirnay et al. (2009), 72.6% isolates from the studies mentioned above (Lanotte et al. of all P. aeruginosa isolates harbored the exoS gene, 23.1% 2004, 80%; Feltman et al. 2001, 75%). ExoS is responsible contained the exoU gene and with the exception of three for direct tissue destruction in lung infection and may be strains, the carriage of exoU and exoS was mutually important for bacterial dissemination (Nicas et al. 1985a; b). exclusive. In contrast, Finnan et al. (2004) found 75% In this study, the distribution of the exoU gene was dissemination of both genes in clinical and environmental very heterogeneous. The gene was found predominantly in P. aeruginosa isolates. P. aeruginosa isolates from LRTIs and wounds. Its prevalence in our wound isolates (51.7%) was similar to that Prevalence of the type III effector proteins-encoding genes ascertained by Feltman et al. (2001) in the USA (40.0%). In with respect to the infection localization contrast, Lomholt et al. (2001) reported an absence of the The distribution of virulence genes encoding the type III Table 2 Distribution of the exo gene patterns among nosocomial effector proteins varied in respect to the infection localization P. aeruginosa isolates (n=176) (Table 3). Gene pattern Number (%) of isolates (1) The presence of exoS was the highest among P. aeruginosa isolates from blood (87.5%) and − − + − exoS /exoU /exoT /exoY 4 (2.3) + − + − significantly different only with those obtained from exoS /exoU /exoT /exoY 12 (6.8) in-patients with lower respiratory tract infections − + + − exoS /exoU /exoT /exoY 5 (2.8) (LRTIs) (53.8%)-P<0.02. − − + + exoS /exoU /exoT /exoY 20 (11.4) (2) The exoU frequencies were the most expressive in the + − + + exoS /exoU /exoT /exoY 83 (47.2) wound (51.7%) and LRTIs P. aeruginosa isolates − + + + exoS /exoU /exoT /exoY 37 (21.0) (40.4%). They were significantly higher than those of + + + − exoS /exoU /exoT /exoY 3 (1.7) the isolates from urine (19.4%), P<0.01 and P<0.02, + + + + exoS /exoU /exoT /exoY 12 (6.8) respectively, and blood (12.5%), P<0.01 and P<0.05, respectively. exo Genes encoding type III effector proteins, + presence, − absence Ann Microbiol (2010) 60:503–509 507 Table 3 Prevalence (as percentage) of the type III effector proteins-encoding genes in nosocomial P. aeruginosa isolates, in respect to the infection site Gene Isolate origin Urine (n=67) LRTIs (n=52) URTIs (n=17) Wounds (n=29) Blood (n=8) Total (n=176) exoS 62.7 53.8 70.6 62.1 87.5 61.9 exoU 19.4 40.4 35.3 51.7 12.5 32.4 exoT 100.0 100.0 100.0 100.0 100.0 100.0 exoY 85.1 82.7 94.1 82.8 100.0 85.8 LRTIs Lower respiratory tract infections, URTIs upper respiratory tract infections, exoS exoenzyme S-encoding gene, exoU exoenzyme U-encoding gene, exoT exoenzyme T-encoding gene, exoY exoenzyme Y-encoding gene Frequency of the type III effector gene in all studied isolates of P. aeruginosa, including three bile isolates exoU in a small series of 11 wound isolates of P. aeruginosa to harbor the exoU gene and to display high swimming (Lomholt et al. 2001). Our urine isolates showed a low motility and adhesiveness. percentage of exoU carriers (19.4%), while Lomholt et al. Recently, it was reported the worldwide spread and did not find urine isolates harboring the exoU gene. persistence of MDR clone comprising P. aeruginosa O12 + − It is known that 90% of ExoU-producing P. aeruginosa isolates which exhibited the exoS /exoU genotype (Pirnay strains are associated with severe infections (Hauser et al. et al. 2009). Moreover, most MDR clinical isolates of 2002). Of the type III secretion proteins, ExoU is the P. aeruginosa reveal either O11 or O12 serotype (Sekiguchi most cytotoxic. Its secretion is a marker for highly et al. 2007; Pirnay et al. 2009). Evaluation of the spread of virulent P. aeruginosa isolates obtained from patients with these serotypes among Bulgarian nosocomial MDR hospital-acquired pneumonia (Schulert et al. 2003). P. aeruginosa strains, as well as their corresponding effector genotypes, should be objectives of our future investigations. Prevalence of the type III effector proteins-encoding genes Whether MDR P. aeruginosa strains necessarily express with respect to the antimicrobial resistance a more virulent phenotype continues to remain a contro- versial issue (Di Martino et al. 2002). Of the 176 nosocomial P. aeruginosa isolates, 101 (57.4%) In conclusion, our results suggest that type III secretion were MDR. One of the genes, exoU manifested a genes are found in nearly all investigated P. aeruginosa but significantly higher spread (P<0.001) among MDR than that individual isolates from distinct infection sites differ in in non-MDR strains of P. aeruginosa (42.6 vs 18.7%) their effector genotypes. Our data confirm that the exoU (Fig. 2). Twelve isolates contained the four studied genes (Table 2), moreover 8 of them were MDR. 100.00% In a recent accomplished study, Garey et al. (2008) determined the prevalence of exoU and exoS from blood- 80.00% stream isolates of hospitalized patients with P. aeruginosa bacteremia and ascertained that the isolates containing the 60.00% exoU gene were significantly more resistant (P<0.05) to different classes of antimicrobials: β-lactams (piperacillin/ 40.00% tazobactam, ceftazidime, cefepime, carbapenems), fluoro- quinolones, and aminoglycosides (gentamicin). Other 20.00% research suggested that the multidrug resistance in ocular P. aeruginosa was more commonly associated with strains 0.00% having cytotoxicity and exoU gene, and belonging to exoS exoU exoT exoY serotype E (Zhu et al. 2006). Recently, Zaborina et al. MDR (n=101) non-MDR (n=75) (2006) screened consecutively isolated MDR P. aerugi- nosa clinical strains for their ability to disrupt the integrity Fig. 2 Prevalence (as percentages) of the type III effector proteins- of human cultured intestinal epithelial cells (Caco-2) and encoding genes among MDR and non-MDR P. aeruginosa isolates correlated these findings to related virulence phenotypes from non-cystic fibrosis patients. MDR multidrug-resisrant, exoS such as adhesiveness, motility, biofilm formation and exoenzyme S-encoding gene, exoU exoenzyme U-encoding gene, cytotoxicity. These strains were characterized and found exoT exoenzyme T-encoding gene, exoY exoenzyme Y-encoding gene 508 Ann Microbiol (2010) 60:503–509 and exoS genes are almost mutually exclusive. Nosocomial Goehring UM, Schmidt G, Pedersen KJ, Aktories K, Barbieri JT (1999) The N-terminal domain of Pseudomonas aeruginosa isolates of P. aeruginosa harboring the exoU gene were exoenzyme S is a GTPase-activating protein for Rho GTPases. more likely to be resistant to multiple antibiotics. The J Biol Chem 274:36369–36372 ubiquity of type III effector proteins-encoding genes among Hauser A (2009) The type III secretion system of Pseudomonas clinical isolates is consistent with an important role for this aeruginosa: infection by injection. Nat Rev Microbiol 7:654–665 Hauser AR, Cobb E, Bodi M, Mariscal D, Valles J, Engel JN, system in human disease. Future studies to determine the Rello J (2002) Type III protein secretion is associated with prevalence of clinical isolates capable of secreting specific poor clinical outcomes in patients with ventilator-associated effector proteins and whether secretion correlates with pneumonia caused by Pseudomonas aeruginosa.CritCare Med 30:521–528 infection prognosis are necessary for evaluation of the type Kirikae T, Mizuguchi Y, Arakawa Y (2008) Investigation of isolation III secretion system in P. aeruginosa pathogenesis. rates of Pseudomonas aeruginosa with and without multidrug resistance in medical facilities and clinical laboratories in Japan. J Acknowledgement This work was supported by a grant from the Antimicrob Chemother 61:612–615 Medical University of Sofia (Council of Medical Science, project no. Krall R, Schmidt G, Aktories K, Barbieri JT (2000) Pseudomonas 4/2009, grant no. 20/2009). aeruginosa ExoT is a Rho GTPase-activating protein. Infect The authors declare that they have no conflict of interest. Immun 68:6066–6068 Kurahashi K, Kajikawa O, Sawa T, Ohara M, Gropper MA, Frank DW, Martin TR, Wiener-Kronish JP (1999) Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J Clin References Invest 104:743–750 Lanotte P, Watt S, Mereghetti L, Dartiguelongue N, Rastegar-Lari A, Allewelt M, Coleman FT, Grout M, Priebe GP, Pier GB (2000) Goudeau A, Quentin R (2004) Genetic feature of Pseudomonas Acquisition of expression of the Pseudomonas aeruginosa ExoU aeruginosa isolates from cystic fibrosis patients compared with cytotoxin leads to increased bacterial virulence in a murine model those of isolates from other origins. J Med Microbiol 53:73–81 of acute pneumonia and systemic spread. Infect Immun 68:3998– Lomholt JA, Poulsen K, Kilian M (2001) Epidemic population 4004 structure of Pseudomonas aeruginosa: evidence for a clone that Berthelot P, Attree I, Plésiat P, Chabert J, de Bentzmann S, Pozzetto B, is pathogenic to the eye and that has a distinct combination of Grattard F, the Groupe d’Etudes des Septicémies à Pseudomonas virulence factors. Infect Immun 69:6284–6295 aeruginosa (2003) Genotypic and phenotypic analysis of type III Nicas TI, Bradley J, Lochner JE, Iglewski BH (1985a) The role of secretion system in a cohort of Pseudomonas aeruginosa exoenzyme S in infections with Pseudomonas aeruginosa.J bacteremia isolates: evidence for a possible association between Infect Dis 152:716–721 O serotypes and exo genes. J Infect Dis 188:512–518 Nicas TI, Frank DW, Stenzel P, Lile JD, Iglewski BH (1985b) Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. Blanc DS, Petignat C, Janin B, Bille J, Francioli P (1998) Frequency Eur J Clin Microbiol 4:175–179 and molecular diversity of Pseudomonas aeruginosa upon Pagani L, Colinon C, Migliavacca R, Labonia M, Docquier JD, admission and during hospitalization: a prospective epidemiologic Nucleo E, Spalla M, Li Bergoli M, Rossolini GM (2005) study. Clin Microbiol Infect 4:242–247 Nosocomial outbreak caused by multidrug-resistant Pseudomonas CLSI (2007) Performance standards for antimicrobial susceptibility testing, 17th informational supplement, M100-S17. Clinical and aeruginosa producing IMP-13 metallo-β-lactamase. J Clin Laboratory Standards Institute, Wayne Microbiol 43:3824–3828 Di Martino P, Gagniere H, Berry H, Bret L (2002) Antibiotic Phillips RM, Six DA, Dennis EA, Ghosh P (2003) In vivo resistance and virulence properties of Pseudomonas aeruginosa phospholipase activity of the Pseudomonas aeruginosa cytotoxin strains from mechanically ventilated patients with pneumonia in ExoU and protection of mammalian cells with phospholipase A intensive care units: comparison with imipenem-resistant inhibitors. J Biol Chem 278:41326–41332 extra-respiratory tract isolates from uninfected patients. Pinheiro MRS, Lacerda HR, Melo RGL, Maciel MA (2008) Microbes Infect 4:613–620 Pseudomonas aeruginosa infections: factors relating to mortality Engel J, Balachandran P (2009) Role of Pseudomonas aeruginosa with emphasis on resistance pattern and antimicrobial treatment. type III effectors in disease. Curr Opin Microbiol 12:61–66 Braz J Infect Dis 12:509–515 Feltman H, Schulert G, Khan S, Jain M, Peterson L, Hauser AR Pirnay J-P, Bilocq F, Pot B, Cornelis P, Zizi M, Van Eldere J, (2001) Prevalence of type III secretion genes in clinical and Deschaght P, Vaneechoutte M, Jennes S, Pitt T, De Voss D (2009) environmental isolates of Pseudomonas aeruginosa. Microbiology Pseudomonas aeruginosa population structure revisited. PLoS 147:2659–2669 ONE 4:e7740. doi:10.1371/journal.pone.0007740 Finck-Barbancon V, Goranson J, Zhu L, Sawa T, Wiener-Kronish JP, Rajan S, Saiman L (2002) Pulmonary infections in patients with cystic Fleiszig SM, Wu C, Mende-Mueller L, Frank DW (1997) ExoU fibrosis. Semin Respir Infect 17:47–56 expression by Pseudomonas aeruginosa correlates with acute Schulert GS, Feltman H, Rabin SD, Martin CG, Battle SE, Rello J, cytotoxicity and epithelial injury. Mol Microbiol 25:547–557 Hauser AR (2003) Secretion of the toxin ExoU is a marker for Finnan S, Morrissey JP, O’Gara F, Boyd EF (2004) Genome highly virulent Pseudomonas aeruginosa isolates obtained from diversity of Pseudomonas aeruginosa isolates from cystic patients with hospital-acquired pneumonia. J Infect Dis fibrosis patients and the hospital environment. J Clin Microbiol 188:1695–1706 42:5783–5792 Sekiguchi J, Asagi T, Miyoshi-Akiyama T, Kasai A, Mizuguchi Y, Araake M, Fujino T, Kikuchi H, Sasaki S, Watari H, Kojima T, Garey KW, Vo QP, Larocco MT, Gentry LO, Tam VH (2008) Miki H, Kanemitsu K, Kunishima H, Kikuchi Y, Kaku M, Prevalence of type III secretion protein exoenzymes and Yoshikura H, Kuratsuji T, Kirikae T (2007) Outbreaks of antimicrobial susceptibility patterns from bloodstream isolates multidrug-resistant Pseudomonas aeruginosa in community of patients with Pseudomonas aeruginosa bacteremia. J hospitals in Japan. J Clin Microbiol 45:979–989 Chemother 20:714–720 Ann Microbiol (2010) 60:503–509 509 Stover CK, Pham XQ, Erwin AL et al (2000) Complete genome hospitalised patients: clinical features, risk-factors and outcomes. sequence of Pseudomonas aeruginosa PAO1, an opportunistic Clin Microbiol Infect 12:63–68 pathogen. Nature 406:959–964 Winstanley C, Kaye SB, Neal TJ, Chilton HJ, Miksch S, Hart CA, Strateva T (2008) Microbiological and molecular-genetic investiga- Microbiology Ophthalmic Group (2005) Genotypic and phenotypic tions on resistance mechanisms and virulence factors in clinical characteristics of Pseudomonas aeruginosa isolates associated with strains of Pseudomonas aeruginosa. PhD dissertation, Medical ulcerative keratitis. J Med Microbiol 54:519–526 University of Sofia, Bulgaria Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW (1998) ExoY, Tamura M, Ajayi T, Allmond LR, Moriyama K, Wiener-Kronish JP, an adenylate cyclase secreted by the Pseudomonas aeruginosa type Sawa T (2004) Lysophospholipase A activity of Pseudomonas III system. Proc Natl Acad Sci USA 95:13899–13904 aeruginosa type III secretory toxin ExoU. Biochem Biophys Res Zaborina O, Kohler JE, Wang Y, Bethel C, Shevchenko O, Wu L, Commun 316:323–331 Turner JR, Alverdy JC (2006) Identification of multi-drug Vallis AJ, Fink-Barbacon V, Yahr TL, Frank DW (1999) Biological resistant Pseudomonas aeruginosa clinical isolates that are effects of Pseudomonas aeruginosa type III-secreted proteins on highly disruptive to the intestinal epithelial barrier. Ann Clin CHO cells. Infect Immun 67:2040–2044 Microbiol Antimicrob 5:14 Van Delden C, Iglewski BH (1998) Cell-to-cell signaling and Zhu H, Conibear TCR, Bandara R, Aliwarga Y, Stapleton F, Willcox Pseudomonas aeruginosa infections. Emerg Infect Dis 4:551– MDP (2006) Type III secretion system-associated toxins, 560 proteases, serotypes, and antibiotic resistance of Pseudomonas Wang CY, Jerng JS, Cheng KY, Lee LN, Yu CJ, Hsueh PR, Yang PC aeruginosa isolates associated with keratitis. Curr Eye Res (2006) Pandrug-resistant Pseudomonas aeruginosa among 31:297–306

Journal

Annals of Microbiology – Springer Journals

Published: Jun 12, 2010

There are no references for this article.