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Int. J. Hyg. Environ. Health 211 (2008) 200–204 www.elsevier.de/ijheh
Are antibiotic-resistant Pseudomonas aeruginosa isolated from hospitalised patients recovered in the hospital efﬂuents? E. Tume´oa, H. Gbaguidi-Haorea, I. Patryb, X. Bertranda, M. Thouvereza, D. Talona, a
Service d’Hygie`ne Hospitalie`re, CHU Jean Minjoz, 25030 Besanc¸on Cedex, France Service de Bacte´riologie, CHU Jean Minjoz, 25030 Besanc¸on Cedex, France
Received 27 July 2006; received in revised form 19 February 2007; accepted 28 February 2007
Abstract Previous reports have studied the presence of antibiotic-resistant Pseudomonas aeruginosa strains in hospital wastewater without determination of their clonal relationship with the clinical strains of this species. The objectives of this study were to quantify the presence of P. aeruginosa in wastewater of our hospital, to determine their antibioticresistance proﬁle and to potentially trace clinical antibiotic-resistant strains from patients to wastewater. Specimens were taken at the end of the wastewater network of our hospital just before the reject in the collective network of the town. Two specimens were taken each Monday during 12 weeks. All P. aeruginosa isolates recovered from hospitalised patients during the study period were collected. Genotyping of both clinical and wastewater isolates was determined by using pulsed-ﬁeld gel electrophoresis (PFGE). The antibiotic-resistance proﬁle of wastewater isolates was different from that of clinical isolates. The mechanisms involved in antibiotic resistance were different according to the origin of the isolates (wastewater versus human isolates). There was no common PFGE pattern in antibiotic-resistant P. aeruginosa from humans and wastewater. This study suggests that the risk of spread of antibiotic resistance in hospital wastewater is limited. r 2007 Elsevier GmbH. All rights reserved. Keywords: Pseudomonas aeruginosa; Hospital wastewater; Antibiotic resistance; PFGE
Introduction Pseudomonas aeruginosa is a ubiquitous organism, able to persist in a large number of environments. This is due to its ability to utilise different organic compounds as energy sources and to survive for longer periods as long as sufﬁcient moisture is available. The natural and permanent reservoir of this hydrophilic microorganism therefore consists of environmental polymicrobial societies independent from humans. P. aeruginosa is a Corresponding author. Tel.: +3 81 66 82 86; fax: +3 81 66 89 14.
E-mail address: [email protected]
(D. Talon). 1438-4639/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2007.02.010
common hospital-acquired pathogen of the respiratory tract and urinary tract, in all departments of the hospital but especially in intensive care units (ICU) (Jarvis and Martone, 1992; Spencer, 1996). The incidence of nosocomial P. aeruginosa infections has increased in recent decades (Navon-Venezia et al., 2005). This is partly due to the increase in the number of patients prone to such infection. High mortality and morbidity rates have been observed for P aeruginosa infections, especially in cases of respiratory tract infection (Bertrand et al., 2001; Fagon et al., 1996). Moreover, P. aeruginosa expresses natural resistance to many antibiotics and has the capacity to acquire many mechanisms of resistance (Hocquet et al., 2003).
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Studies on the bacterial contamination of hospital wastewater have included this specie as well as other classical indicators (Chitnis et al., 2004; Tsai et al., 1998). Various biological ﬂuids from patients colonised or infected by P. aeruginosa are rejected in the wastewater network of the hospital and could contaminate the environment. The dissemination of antibiotic-resistant strains in this way could potentially represent a risk of public health, this specie being well adapted to survive and multiply in this environment. The published studies on this topic report quantitative analysis of this microorganism in hospital wastewater and determination of antibiotic-resistance proﬁle (Chitnis et al., 2004; Tsai et al., 1998). To our knowledge, no study has attempted to compare the molecular proﬁle of wastewater and clinical isolates of P. aeruginosa. Our objectives were to quantify the presence of P. aeruginosa in the wastewater of our hospital, to determine their antibiotic-resistance proﬁle and to trace the clinical multi-resistant strains.
Material and methods Study setting Besanc¸on Hospital is a 1205-bed acute-care university-afﬁliated hospital. The water consumption was 480 l per bed and per day in 2004. There was only one point of rejection of hospital wastewater in the general wastewater network of the town. The wastewater of a non-hospital building (community building), located in another part of the town, was used as control.
Wastewater sampling Wastewater samplings were performed at the end of the hospital and community-building networks. During 12 weeks (from March to May 2005), two specimens of 100 ml were collected each Monday (one at 9 AM and one at 2 PM) in sterile glass bottles.
Bacteriological analysis One hundred ml were diluted in 0.5 ml of serum saline and plated onto cetrimide agar broth (Bio-Rad, Ivry sur Seine, France). The plates were incubated at 41 1C for 48 h. Presumptive identiﬁcation of P. aeruginosa was performed on each CFU and was based on standard procedures (production of pyoverdine, and positive oxydase test) and conﬁrmed using biochemical API 20 NE system (BioMe´rieux, Marcy l’e´toile, France). Based on the morphological aspect, a maximum of 5 CFUs per plate (5 CFU if more than 5 CFU on the plate) were isolated and considered for antibiotic-susceptibility testing. Sixteen antibiotics were tested against each
isolate. Concomitantly, all the non-duplicate isolates of P. aeruginosa recovered from clinical specimens of hospitalised patients were collected. Antimicrobial susceptibility of all the isolates (environmental and clinical) was determined by using the disc diffusion method as recommended by the Antibiogram Committee of the French Society for Microbiology (Soussy et al., 2000). We also deﬁned ﬁve antibiotypes: wild type (susceptible to ticarcillin, gentamicin, ciproﬂoxacin and imipenem), isolated overproduction of an efﬂux system (intermediate to ticarcillin, susceptible to piperacillin and negative nitroceﬁn test), enzymatic resistance to b-lactams (resistant to ticarcillin and positive nitroceﬁn test), resistance to ﬂuoroquinolones (non-susceptible to ﬂuoroquinolones) and multi-drug resistance (non-susceptible to ceftazidime, gentamicin, imipenem and ciproﬂoxacin).
Genotyping The genetic similarity of strains was investigated by pulsed-ﬁeld gel electrophoresis (PFGE) (CHEF DRIII, Bio-Rad, Ivry sur Seine, France) using DraI (Boehringer Mannheim, Germany) as previously described (Talon et al., 1995). Samples of SmaI-restricted DNA of Staphylococcus aureus NCTC 8325 were included in each run as an internal reference. The banding patterns were analysed by scanning photographic negatives. GelCompar software was used for cluster analysis (Applied Maths, Kortrijk, Belgium). Each strain was ﬁrst compared with all other strains and the Dice correlation coefﬁcient was used to calculate similarity. The strains were then grouped and the UPGMA clustering algorithm was used to depict the groups as a dendrogram. Major restriction patterns were deﬁned as those differing by more than three fragments, with a similarity index o85%, as described by Tenover et al. (1995).
Statistical analysis Data analysis was performed using Epi-Info software (version 2002; Centers for Disease Control and Prevention, Atlanta, GA) and SAS software (version 8.0; SAS Institute, Inc., Cary, NC). Categorical variables were compared using Pearson’s Chi-square test or Fisher’s exact test, as appropriate. Continuous variables were compared using Student’s t-test or Mann–Whitney’s U-test. All tests were two-tailed, and a p-value of less than 0.05 was considered statistically signiﬁcant.
Results Nineteen of 20 (95%) and 14 of 20 (70%) samples were positive with P. aeruginosa in the hospital and
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community-building wastewater, respectively (Table 1). A total of 77 isolates were recovered: 44 in the hospital wastewater and 33 in the community-building wastewater. All the isolates recovered in the wastewater of the community-building had a wild-type resistance pattern. The distribution of the antibiotypes is reported in Table 1. During the 12-weeks study period, 183 nonduplicate isolates were recovered from hospitalised patients. The frequency of resistance to ﬂuoroquinolones was higher among patients’ isolates than among hospital wastewater isolates (p ¼ 0.004). On the other hand, the overexpression of an efﬂux system was higher in wastewater isolates (p ¼ 0.015) (Table 1). Twenty multi-drug-resistant isolates were genotyped: six from hospital wastewater and 14 from patients. Genotyping displayed 14 pulsotypes and none of the environmental isolates clustered in the same PFGE pattern than the patients’ isolates (Fig. 1). The two wastewater isolates
Discussion Our results show that there is a difference regarding the frequency of antibiotic resistance among strains of P. aeruginosa isolated from community-building and hospital wastewaters, the occurrence of resistant strains being much more frequent in the hospital setting. Moreover, based on phenotypic data, our study suggests that, in our hospital, the mechanisms of antibiotic resistance are different according to whether P. aeruginosa isolates are environmental or clinical. For instance, ﬂuoroquinolone resistance, which is mainly acquired by mutation and selection as a result of ﬂuoroquinolone exposure (Hansen et al., 2006), is more frequent in
Frequency of various antibiotypes of P. aeruginosa isolated in Besanc¸on Hospital and community-building wastewater
Wild-type Overexpression of efﬂux system Production of b-lactamase Fluoroquinolone resistance Multi-drug resistance a
belonging to the same pulsotype C were isolated in two samples taken in 2 consecutive weeks.
Community-building (n ¼ 33)
Hospital (n ¼ 44)
Hospital (n ¼ 183)
100% (n ¼ 33) 0% 0% 0% 0%
34.1% (n ¼ 15) 25% (n ¼ 11) 20.4% (n ¼ 9) 9.1% (n ¼ 4) 13.6% (n ¼ 6)
o 105 0.0055 0.016 0.21 0.075
42.6% (n ¼ 78) 10.9% (n ¼ 20) 26.2% (n ¼ 48) 36.6% (n ¼ 67) 9.3% (n ¼ 17)
0.30 0.015 0.43 0.0004 0.89
Comparison of the frequency of resistance between hospital wastewater and patient isolates of P. aeruginosa.
Fig. 1. Dendrogram of percentage of similarity between macrorestriction (Dra I) DNA pattern of isolates of multi-drug resistant P. aeruginosa from patients and efﬂuents.
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clinical isolates (36.6%) than in wastewater isolates (9.1%). Conversely, overproduction of efﬂux systems appears to be more frequent in wastewater isolates (25.0%) than in clinical isolates (10.9%). These data have to be conﬁrmed with a measure of the genetic expression of efﬂux genes by using RT-PCR and sequencing of regulator genes (Deplano et al., 2005; Hocquet et al., 2003) but the phenotypic determination of efﬂux pump overexpression is well correlated with efﬂux pump gene overexpression (Hocquet et al., 2003). These differences may reﬂect different types of exposure. For clinical isolates, antibiotic exposure is recognised as the main risk factor for acquisition of ﬂuoroquinolone and b-lactam resistance (Carmeli et al., 1999; Harris et al., 1999). On the other hand, disinfectants such as triclosan and quaternary ammonium, which are extensively used in hospitals, are substrates of efﬂux pump systems of P. aeruginosa (Chuanchuen et al., 2002, 2003; Walsh et al., 2003). These efﬂux pumps are now recognised to play a major role in non-enzymatic mechanisms of acquired resistance in P. aeruginosa. As a consequence of the extensive use of disinfectants in hospital, these products are in high concentration in hospital efﬂuent and may exert a selective pressure on P. aeruginosa strains. Spread of multi-drug-resistant bacteria through hospital wastewater is a cause of concern, especially for hydrophilic species such as P. aeruginosa for which the hospital wastewater environment may be an ideal habitat. To our knowledge, our study is the ﬁrst to trace the clinical strains of P. aeruginosa in hospital efﬂuent, by using PFGE, a technique considered to be the most accurate genotyping method of P. aeruginosa for epidemiological purposes (Speijer et al., 1999). Surprisingly, none of the clinical MDR strains were recovered in efﬂuents. The number of typed strains is limited and our ﬁndings should be considered carefully. However, our results are consistent with those of Valles et al. (2004) who found that strains present in tap water of ICU were rarely associated with human infections. It is possible that in hospital, P. aeruginosa strains are clustered in two distinct genetic groups according to their origin: environmental strains including tap water and wastewater isolates and strains causing invasive hospital infection. These data should be conﬁrmed by additional research in a large-scale prospective study including virulence assays (Fenner et al., 2006) and population genetic analysis using multi-locus sequence typing (Vernez et al., 2005).
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