Mobile genetic elements of Pseudomonas aeruginosa isolates from hydrotherapy facility and respiratory infections

Authors

  • S. G. Pereira,

    1. Center for Pharmaceutical Studies, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
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  • O. Cardoso

    Corresponding author
    1. Center for Pharmaceutical Studies, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
    • Corresponding author: O. Cardoso, Faculty of Pharmacy of University of Coimbra, Health Sciences Campus, Azinhaga de Santa Comba, 3004–568 Coimbra, Portugal

      E-mail: ocardoso@ci.uc.pt

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Abstract

The content of mobile genetic elements in Pseudomonas aeruginosa isolates of a pristine natural mineral water system associated with healthcare was compared with clinical isolates from respiratory infections. One isolate, from the therapy pool circuit, presented a class 1 integron, with 100% similarity to a class 1 integron contained in plasmid p4800 of the Klebsiella pneumoniae Kp4800 strain, which is the first time it has been reported in P. aeruginosa. Class 1 integrons were found in 25.6% of the clinical isolates. PAGI1 orf3 was more prevalent in environmental isolates, while PAGI2 c105 and PAGI3 sg100 were more prevalent in clinical isolates. Plasmids were not observed in either population.

Pseudomonas aeruginosa is known for its environmental ubiquity and its ability to cause several infections in immunocompromised humans, with a high impact on morbidity and mortality. Ten per cent of its genome is flexible, and limited to specific chromosomal locations referred to as genomic islands (GEIs). PAGI (P. aeruginosa genomic island) 1 was the first GEI described in P. aeruginosa, in an isolate from a urinary infection sample, and is present in about 85% of all clinical isolates. PAGI2 was first described in a cystic fibrosis patient and PAGI3 in an aquatic isolate. PAGI4 to 11 and PAPI (P. aeruginosa pathogenic island) 1 and 2 have been reported, but less frequently [1]. Integrons and plasmids are also part of the P. aeruginosa flexible genome and all are considered mobile genetic elements (MGEs) [2], contributing to the success of the niche-based adaptation of the strains and spread of antibiotic resistance genes [3]. Natural mineral water (NMW) is used in hydrotherapy facilities, commonly named thermae, to treat mainly respiratory and rheumatological conditions, considered risk factors for pneumonia [4]. Regulations prohibit disinfection of NMW [5], which may lead to thermae users being exposed to contaminated NMW during treatment. Some studies report MGEs in P. aeruginosa from water [6-9], but none reports them in NMW from thermae settings. These facilities are relevant to public health because they provide medical healthcare treatments.

Ninety non-clonal P. aeruginosa isolates were used: 51 thermae (planktonic and biofilm) isolates and 39 (respiratory infections) clinical isolates, previously selected by pulsed-field gel electrophoresis (PFGE) (Table 1). All isolates were screened for plasmid content, class 1, 2 and 3 integrons and PAGI1, 2 and 3. Conventional plasmids were screened by the alkaline lysis method [10]. Megaplasmids were detected by S1-digestion PFGE [11]. Integrons were screened using a two-step strategy: first, screening of intI1, intI2 and intI3 integrases [12], and then integron content screening, with adequate primers [13]. PAGI1 was screened using orf3, orf18 and orf42 primers, PAGI2 using c22 and c105 primers and PAGI3 using sg8 and sg100 primers [14]. All positive PCR-amplicons were sequenced and compared with previously reported sequences, available in GenBank®. Chi-square tests (95% significance level) were used to infer the distribution of MGEs between populations.

Table 1. Description of isolates and respective MGE profile. Isolates were grouped according to presence/absence of MGE and not taking into account the integron contents of each isolate
Isolate numberTypeLocationClass1 integronPAG1PAGI2PAGI3MGE type
orf3 orf18 orf42 c22 c105 sg8 sg100
  1. Clin, clinical isolate; Sput, sputum; Aspir, aspirate; NMW, natural mineral water; Plank, planktonic isolate; Biofilm, biofilm isolate; Ir, irrigation; Nebul, nebulizer; ICU, intensive care unit; IDU, infectious diseases unit; equip, equipment.

071Clin – SputUnit 2+++++MGE-1
133Clin – SputNeurology++++MGE-2
097Clin – AspirOutpatient++++MGE-3
159Clin – SputEmergency++++
182Clin – SputPneumology++++
198Clin – AspirMedicine++++
F42NMW PlankBorehole 2+++MGE-4
C104NMW PlankTherapy pool+++
C270NMW PlankHydromassage+++
C331NMW PlankNasal Ir./Aerosol+++
B14NMW BiofilmCollective nebul.+++
B15NMW BiofilmCollective nebul.+++
B19NMW BiofilmCollective nebul.+++
B26NMW BiofilmCollective nebul.+++
003Clin – AspirICU+++
079Clin – AspirMedicine+++
107Clin – SputMedicine+++
111Clin – SputNeurology+++
125Clin – AspirICU+++
209Clin – SputPneumology+++
B52NMW BiofilmCollective nebul.+++MGE-5
043Clin – AspirICU+++MGE-6
204Clin – SputIDU++++MGE-7
104Clin – AspirPneumology+++MGE-8
051Clin – SputPneumology+++MGE-9
080Clin – SputOutpatient+++
109Clin – AspirDay Care+++
F44NMW PlankBorehole 1++MGE-10
C9NMW PlankVichy shower++
C17NMW PlankMain deposit++
C34NMW PlankNasal Ir./Aerosol++
C84NMW PlankDrinking++
C187NMW PlankMain deposit++
C230NMW PlankHydromassage++
B1NMW BiofilmCollective nebul.++
B21NMW BiofilmAerosol equip.++
B29NMW BiofilmAerosol equip.++
B30NMW BiofilmAerosol equip.++
B32NMW BiofilmCollective nebul.++
B75NMW BiofilmCollective nebul.++
B94NMW BiofilmAerosol equip.++
B95NMW BiofilmAerosol equip.++
062Clin – SputNeuro-surgery++
129Clin – SputNeurology++
137Clin – AspirUnit 2++
B39NMW BiofilmCollective nebul.++MGE-11
B63NMW BiofilmCollective nebul.++
095Clin – AspirMedicine++MGE-12
158Clin – AspirICU++
B110NMW BiofilmAerosol equip.++
B86NMW BiofilmAerosol equip.++MGE-13
B20NMW BiofilmCollective nebul.++MGE-14
F1NMW PlankBorehole 2+MGE-15
F14NMW PlankBorehole 1+
F29NMW PlankBorehole 2+
F34NMW PlankBorehole 2+
F49NMW PlankBorehole 1+
F61NMW PlankBorehole 1+
F65NMW PlankBorehole 2+
C1NMW PlankNasal Ir./Aerosol+
C19NMW PlankHydromassage+
C30NMW PlankTherapy pool+
C97NMW PlankVichy shower+
C172NMW PlankHydromassage+
C176NMW PlankPharinx shower+
C301NMW PlankTherapy pool+
B12NMW BiofilmCollective nebul.+
B40NMW BiofilmCollective nebul.+
B79NMW BiofilmAerosol equip.+
B107NMW BiofilmCollective nebul.+
B108NMW BiofilmAerosol equip.+
214Clin – SputNeuro-surgery+
115Clin – SputPneumology++MGE-16
012Clin – SputNeurology+MGE-17
029Clin – SputNeurology+
058Clin – AspirNeuro-surgery+
067Clin – AspirMedicine+
076Clin – SputMedicine+
135Clin – AspirICU+MGE-18
C268NMW PlankTherapy pool+MGE-19
C101NMW PlankTherapy poolMGE-20
B65NMW BiofilmCollective nebul.
013Clin – SputNeurology
030Clin – SputNeurology
061Clin – SputNeurology
103Clin – SputNeuro-surgery
127Clin – SputIDU
142Clin – SputNeurology
218Clin – AspirIDU
223Clin – SputIDU

The Pseudomonas aeruginosa flexible genome is important for it to thrive in diverse environments [1, 2], conferring specific phenotypes that are advantageous under selective pressure, particularly drug-resistance and virulence features. Class 1 integron is frequently found in P. aeruginosa clinical isolates, class 2 is rare and class 3 has not yet been reported [3]. Integrons were not detected in thermae isolates, except in one isolate from the therapy pool circuit, where contamination by pool users may occur. The isolate contained a class1-integron 800 bp PCR-amplicon, 100% similar to Klebsiella pneumoniae strain Kp 4800 plasmid p4800 class 1 integron (EU622041.1), this being the first description in P. aeruginosa (Gen Bank accession number KF525705) and suggesting horizontal gene transfer between the two species. Rosser and Young [6] reported 3.6% class 1 integrons in a 3000 bacterial population from an estuary. Zanetti et al. [9] reported plasmids and class 1 and 2 integrons in superficial water P. aeruginosa isolates. Both systems are subjected to pollution, including antibiotics [15], in contrast to NMW, wholesome water that cannot suffer any chemical or microbiological change prior to use [5]. Ten clinical isolates presented class 1 integrons. The majority contained aadA11 and half had aadB gene cassettes. Ruiz-Martínez et al. [8] presented similar results, but aadB was the most frequent gene cassette, followed by aadA11. Class 2 and 3 integrons and plasmids were not detected in the present study, as reported in the literature [3, 8].

Regarding GEIs, PAGI1 orf3 and orf18 and PAGI2 c105 were the only PAGI genes present in both populations and were those with a higher prevalence (Fig. 1). PAGI1 orf3 was more prevalent in thermae (p 0.001). PAGI2 c105 (p 0.035) and PAGI3 sg100 (p 0.013) were more prevalent in clinical isolates. Several studies report GEIs in strains from human infections and aquatic habitats [1, 7, 14, 16], consistent with the fact that some GEIs encoded functions contribute to general adaptability and competitiveness rather than exclusively to infection processes [16], and virulence traits are probably natural features that bacteria have in order to avoid predation and survive in soil and water [17, 18], which may explain the presence of GEIs in thermae isolates.

When considering the MGE profile of each isolate, it was possible to observe that clinical isolates presented higher MGE diversity (15 MGE-types) and higher content of GEIs per isolate (MGE-type 1 to 4) than environmental ones (Table 1). MGE-15 (presence of orf3) was more frequent (22.2%), followed by MGE-10 (presence of orf3 and orf18) (20%), both mainly represented by NMW isolates. Clinical isolates presented higher frequency of isolates with no MGEs (n = 9), while only two NMW isolates had no MGEs. This is consistent with a more homogeneous distribution of MGEs, mainly GEIs, in the NMW environment than in the clinical setting, probably due to less stressful genetic conditions than those found in the hospital setting, filled with antibiotics, antiseptics and disinfectants, which may lead to higher gene mobilization between strains, as suggested by Ruiz-Martínez et al. [8].

Figure 1.

Prevalence (%) of PAGI1 (orf3, orf18 and orf42), PAGI2 (c22 and c105) and PAGI3 (sg8 and sg100) genes in natural mineral water and clinical isolates.

This study has first demonstrated the MGE content of pristine NMW P. aeruginosa isolates, almost exclusively restricted to GEIs. A class 1 integron, from K. pneumoniae, was described for the first time in P. aeruginosa, from a NMW therapy pool. This observation, although restricted to one isolate, is worrying because the integron can spread to other bacteria and disseminate to the water system. We think it is important to regularly assess MGEs in thermae facilities, to better understand possible resistance dissemination in these healthcare-associated environments.

Acknowledgements

This study was supported by a doctoral fellowship (SFRH/BD/46668/2008) of the Portuguese Foundation for Science and Technology to Sónia Gonçalves Pereira. The Center for Pharmaceutical Studies financially supported part of the work. The authors wish to thank Centro Hospitalar de Coimbra for providing the clinical isolates included in this study.

Transparency Declaration

The Authors declare no conflicts of interest.

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