Leakage into Portuguese aquatic environments of extended-spectrum- -lactamase-producing Enterobacteriaceae

Sir, Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been recognized as significant nosocomial pathogens during recent decades.1 The spread of these organisms is worrisome since they might also transfer antibiotic resistance genes among different hosts and environments. In this work, we describe the occurrence of ESBLs and integrons among Enterobacteriaceae from Portuguese aquatic environments and analyse the clonal relationship between Portuguese ESBL-producing isolates of environmental, human and animal origin. We identified 16 ESBL-producing Enterobacteriaceae (11 Klebsiella pneumoniae, 4 Escherichia coli and 1 Enterobacter aerogenes) in eight environmental samples collected in Portugal in order to detect cephalosporin-resistant Gram-negative bacteria. The samples included: (i) raw urban wastewater directly from sewer lines downstream of four hospitals located in the Porto area (n = 5; 2001–02); (ii) river water from river Sousa in the north of Portugal (n = 1; 2003); and (iii) Porto marine water (n = 2; 2003–04). River and marine waters were collected close to clandestine discharge points of water streams contaminated by faecal coliforms. Samples were collected in sterile bottles and processed on the same day of collection. River or marine samples were submitted to a vacuum membrane filtration procedure [100 mL on 0.45 µm pore-size membranes (Millipore Corporation, Molsheim, France)] and filters were placed on MacConkey agar plates supplemented with ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Wastewater samples were directly spread on these plates (0.1 mL). ESBL characterization was accomplished by the standard double-disc synergy test, amplification of known bla genes and sequencing.2 Bacterial identification, antibiotic susceptibility tests, conjugation assays and plasmid analysis were performed as described previously.2,3 Clonal relatedness was established by randomly amplified polymorphic DNA analysis (RAPD)-PCR and/or PFGE.3,4 The clonal relationship between environmental strains from this study and ESBL-producing strains widely spread in both hospital (northern and central regions, 2002–04) and community Portuguese settings (northern region, 2003–05) was determined.2,4E. coli phylogenetic groups were identified by multiplex PCR.3 Class 1, 2 and 3 integrons were characterized by PCR-RFLP and sequencing.3 The epidemiological characteristics of the ESBL-producing isolates studied are shown in Table 1, which includes typing results and patterns of resistance to non-β-lactam antibiotics. A diversity of ESBLs was identified from five hospital sewage samples and comprised TEM-116 from different K. pneumoniae strains collected in all five hospital wastewaters, SHV-12 or TEM-10/SHV-27 from K. pneumoniae and TEM-52 from E. coli, each collected from different hospital wastewater streams. From the River Sousa, we identified a TEM-24-producing E. aerogenes strain and a TEM-52-producing E. coli. CTX-M-14 and CTX-M-32 producers, which are commonly recovered from community and hospital settings in south Europe,1 were also identified in two marine coastal waters. All but TEM-116, previously detected from Ria de Aveiro (central Portugal),5 have not been previously recovered from environmental samples. K. pneumoniae was the species most commonly found as an ESBL producer (69%, 11/16). Characteristics of ESBL-producing isolates recovered from river, marine coastal waters and hospital wastewaters from Portugal Patterns transferred by conjugation are underlined. ND, not done; NT, not typeable; neg, strains showing a negative result when amplifying the variable region of class 1 or 2 integrons; PR, private hospital; P1, public hospital 1; P2, public hospital 2; P3, public hospital 3; PhG, phylogenetic group; Gm, gentamicin; Tb, tobramycin; Ak, amikacin; Sm, streptomycin; Sp, spectinomycin; Nt, netilmicin; Km, kanamycin; Su, sulphonamide; Tp, trimethoprim; Te, tetracycline; Cm, chloramphenicol; Cp, ciprofloxacin; Na, nalidixic acid. aStrains showing characteristics of clinical origin. bStrains clonally related to ESBL producers previously recovered from Portuguese hospitals.2 cThis strain also harboured blaVIM-2. Characteristics of ESBL-producing isolates recovered from river, marine coastal waters and hospital wastewaters from Portugal Patterns transferred by conjugation are underlined. ND, not done; NT, not typeable; neg, strains showing a negative result when amplifying the variable region of class 1 or 2 integrons; PR, private hospital; P1, public hospital 1; P2, public hospital 2; P3, public hospital 3; PhG, phylogenetic group; Gm, gentamicin; Tb, tobramycin; Ak, amikacin; Sm, streptomycin; Sp, spectinomycin; Nt, netilmicin; Km, kanamycin; Su, sulphonamide; Tp, trimethoprim; Te, tetracycline; Cm, chloramphenicol; Cp, ciprofloxacin; Na, nalidixic acid. aStrains showing characteristics of clinical origin. bStrains clonally related to ESBL producers previously recovered from Portuguese hospitals.2 cThis strain also harboured blaVIM-2. Several RAPD or PFGE patterns were identified although some clones were persistently recovered, such as TEM-116-producing K. pneumoniae RAPD-type KP-2, isolated in 2001 and 2002, or were clonally related to those previously identified among clinical samples, such as the TEM-24-producing E. aerogenes,2 the TEM-116-producing K. pneumoniae RAPD-type KP-2 and the SHV-12-producing K. pneumoniae RAPD-type KP-5.2 No clonal relationships were found between ESBL-producing Enterobacteriaceae of aquatic and animal origin.4 Two ESBL-producing E. coli belonged to phylogroup D (one CTX-M-14 and one TEM-52-producing isolate). The remaining two isolates belonged to phylogroup A or B1. The blaESBL genes were successfully transferred by conjugation from 31% (5/16) of the strains (Table 1). Although the CTX-M-14-producing E. coli recovered from coastal waters was clonally unrelated to other CTX-M-14-producing strains from Portuguese hospitals or healthy volunteers,2 a plasmid of ca. 80 kb bearing blaCTX-M-14 and showing a similar HpaI digestion pattern was detected in strains from the three origins.2 Most of the studied strains contained intI1 and/or intI2 (n = 14/16 isolates), although variable regions corresponding to class 1 or 2 integrons were only detected in five isolates. The gene cassette arrays of the three class 1 integrons identified were aacA4, which was associated with TEM-24 producers,3aadA2, which was linked to a TEM-116-producing K. pneumoniae strain, and aacA7-blaVIM-2, recovered from a K. pneumoniae isolate also harbouring blaTEM-10 and blaSHV-27. This gene cassette array is frequent among Enterobacteriaceae and Pseudomonas aeruginosa from different countries and has been recovered from environmental metallo-β-lactamase (MBL)-producing P. aeruginosa in Portugal suggesting that this genetic element is widespread.6 The two class 2 integron types characterized were dfrA1-sat2-aadA1-orfX (from CTX-M-14-producing E. coli) and dfrA1-sat2 (from one TEM-52-producing E. coli and one TEM-116-producing K. pneumoniae). The dfrA1-sat2 gene cassette array has not been previously linked to a class 2 integron and it might reflect a deletion from the element mentioned above as a result of different recombinatorial events. Simultaneous conjugative transfer of integrons and ESBL genes was only detected for the K. pneumoniae strain harbouring blaTEM-10, blaSHV-27 and blaVIM-2. The identification in aquatic environments of strains and/or genetic elements associated with ESBLs and MBLs widely disseminated in Portuguese hospitals is of concern and suggests environmental contamination from hospital wastewaters, although a community origin cannot be discarded. Our results underscore the importance of the aquatic environment for maintenance of multiresistant pathogens. This project was funded by REQUIMTE. E. M. was supported by a fellowship from Fundação para a Ciência e a Tecnologia de Portugal (SFRH/BD/11304/2002). No conflicts of interest to declare. We are very grateful to SMAS Porto, Divisão de Controlo e Qualidade and Departamento de Águas Residuais, for collection of wastewater samples.