ABSTRACT
Carbapenemase-producing bacteria cause difficult-to-treat infections related to increased mortality in health care settings. Their occurrence has been reported in raw sewage, sewage-impacted rivers, and polluted coastal waters, which may indicate their spread to the community. We assessed the variety and concentration of carbapenemase producers in coastal waters with distinct pollution levels for 1 year. We describe various bacterial species producing distinct carbapenemases not only in unsuitable waters but also in waters considered suitable for primary contact.
KEYWORDS: antimicrobial resistance bacteria, β-lactamase, recreational coastal waters, Klebsiella pneumoniae carbapenemase (KPC), environmental resistance dissemination
TEXT
Carbapenemase-producing bacteria are opportunistic, often multidrug-resistant, pathogens that commonly cause hospital-acquired infections associated with increased mortality. The ease of horizontal transfer of the resistance determinants further increases the challenges that carbapenemase producers present for public health (1).
Reports of carbapenemase in nonhuman sources, particularly aquatic sources contaminated with sewage, are increasing worldwide (2–6). Under ideal circumstances, most populations are not in direct contact with sewage or polluted rivers, and thus the risk of exposure to carbapenemase producers is thought to be small. However, sewage-contaminated rivers and urban runoffs frequently flow into coastal waters, where numerous recreational activities take place. On the other hand, reports on occurrences of carbapenemase producers in coastal waters are scarce and focus mainly on specific enzyme distribution in sewage-impacted samples (7–9). Here, we assessed the variety and concentration of carbapenemase-producing bacterial organisms along five urban tourist beaches showing distinct levels of pollution.
Water samples were collected twice every 3 months (14 or 15 days apart) between September 2013 and September 2014, which totaled 10 samples from each beach. Sampling sites (latitude/longitude coordinates) were Botafogo (−22.944434, −43.180056), Flamengo (−22.929164, −43.170845), Copacabana (−22.973853, −43.184573), Ipanema (−22.987266, −43.205129), and Leblon (−22.988770, −43.226624). Two liters of seawater were obtained at 15 cm from the surface in a column depth of 100 cm in amber sterile flasks that were kept on ice until processing, and this was performed no longer than 8 h after sampling. Pure samples (200 μl), 200-μl aliquots of their dilutions (1:20 and 1:50), and Millipore membranes (0.22-μm pore size) used to filter different sample volumes (10, 20, 50, 100, 250, and 500 ml) were cultured in 90-mm plates containing CHROMagar Orientation medium (BD Diagnostics, Le Pont de Claix, France) supplemented with imipenem (1 μg/ml). Plates were incubated aerobically at 35 ± 2°C for 18 to 24 h. A single plate per sample was analyzed, with each showing 20 to 50 CFU. All CFU were subcultured under the same conditions and identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (Bruker Daltonics, Biotyper 3.1), following manufacturer instructions.
We used the multiple-tube fermentation technique to enumerate thermotolerant coliforms and Enterolert to determine total enterococci (IDEXX, Westbrook, ME, USA). According to Brazilian legislation, waters are considered suitable for primary contact when levels of enterococci and thermotolerant coliforms are ≤100 and ≤1,000 MPN (most probable number)/100 ml, respectively, in ≥80% of a set of samples collected during the previous 5 weeks (10). Here, we considered the same parameters to qualify single coastal water samples. Waters from Botafogo, Flamengo, Leblon, Ipanema, and Copacabana were classified as unsuitable for primary contact in 10 (100%), 6 (60%), 5 (50%), 3 (30%), and 2 (20%), respectively, of the samples studied.
A total of 807 Gram-negative bacilli were recovered; 522 were eliminated because they belonged to bacterial genera known to produce intrinsic carbapenemases or they were known to have limited relevance to human health in regard to carbapenem resistance. The remaining 285 isolates comprised our study collection: 122 Acinetobacter spp. (43%), 14 Aeromonas spp. (5%), 12 Pseudomonas spp. (4%), and 137 Enterobacteriaceae (48%). Enterobacteriaceae included 92 Enterobacter spp. (32%), 31 Klebsiella spp. (11%), 8 Serratia spp. (3%), 3 Kluyvera spp. (1%), 2 Escherichia spp. (1%), and 1 Citrobacter sp. (<1%).
Aeromonas spp., Enterobacteriaceae, and Pseudomonas spp. were subjected to the modified Carba NP test (11), whereas Acinetobacter spp. were submitted to the CarbAcineto NP test (12). In isolates showing inconclusive test results, we used spectrophotometric assays to investigate carbapenemase production (13).
A total of 146 isolates (51%) showed positive results for carbapenemase production, of which 78 were recovered from Botafogo (53%), 40 from Flamengo (27%), 17 from Leblon (12%), 9 from Ipanema (6%), and 2 from Copacabana (1%). These sites showed carbapenemase producers in 10 (100%), 10 (100%), 6 (60%), 5 (50%), and 2 (20%) of the studied samples, respectively.
The average concentration of carbapenemase producers was highest in Botafogo (1,646 CFU/100 ml), followed by Leblon (6.42 CFU/100 ml), Flamengo (5.38 CFU/100 ml), Ipanema (4.2 CFU/100 ml), and Copacabana (0.04 CFU/100 ml). Unsuitable water samples showed carbapenemase producers more often than suitable ones (64% versus 36%; P < 0.05), reaching concentrations of 6,500 and 12 CFU/100 ml, respectively. Concentrations of carbapenemase producers according to levels of fecal indicator bacteria in the samples studied are depicted in Fig. 1. According to our findings, the breakpoint for thermotolerant coliform counts does not predict absence of carbapenemase producers in suitable waters. Therefore, fecal indicator bacteria counts should be accompanied by a specific test to detect carbapenemase producers, if future studies indicate that such occurrence is an adequate additional parameter to assess the safety of coastal waters for recreational practices.
FIG 1.
Scatterplots correlating the amount of carbapenemase producers with measures of fecal indicator bacteria in enterococci (Spearman's rho = 0.64; P < 0.001) (a) and thermotolerant coliforms (Spearman's rho = 0.80; P < 0.001) (b). Dashed lines, current standards for microbiological quality of coastal water samples according to Brazilian legislation (suitable for primary contact when ≤100 MPN/100 ml enterococci and/or ≤1,000 MPN/100 ml thermotolerant coliforms).
Presence of the carbapenemase-encoding genes blaKPC, blaBKC, blaGES, blaOXA-48, blaNDM, blaSPM, blaIMP, blaVIM, blaSIM, and blaGIM was assessed by PCR followed by amplicon sequencing in all isolates showing carbapenemase production. Acinetobacter spp.-related carbapenem-hydrolyzing class D β-lactamases (CHDLs) (blaOXA-23, blaOXA-24/40, blaOXA-51, blaOXA-58, and blaOXA-143) were also investigated, with primers and cycling conditions described previously (14–20).
All isolates carried at least one of the resistance genes surveyed. The most frequently found gene was blaKPC (100), followed by Acinetobacter spp.-related CHDLs (78), blaGES (10), blaIMP (9), blaVIM (2), blaNDM (1), blaSPM (1), and blaOXA-370 (1). Table 1 shows the occurrence of carbapenemase-producing genes found in isolates studied according to conditions of the water for recreation.
TABLE 1.
Carbapenemase producers and carbapenemase-encoding genes found in Rio de Janeiro's coastal waters between September 2013 and September 2014, according to beach water quality for primary contact
| Carbapenemase-producing isolate (n) | Resistance determinants of bacterial isolates (n) from: |
|
|---|---|---|
| Suitable watera | Unsuitable water | |
| Acinetobacter spp. (38) | blaIMP-16 + blaOXA-23 + blaOXA-58 + blaOXA-143 (1) | blaIMP-16 + blaOXA-58 + blaOXA-143 + blaGES-16 (1) |
| blaIMP-16 + blaOXA-58 + blaOXA-143 (2) | blaOXA-51 + blaOXA-23 + blaOXA-143 (9) | |
| blaOXA-51 + blaOXA-23 + blaOXA-143 (1) | blaOXA-58 + blaOXA-24/40 + blaOXA-143 (2) | |
| blaIMP-1 + blaOXA-58 + blaOXA-143 (1) | blaIMP-16 + blaOXA-58 + blaOXA-143 (1) | |
| blaOXA-24/40 + blaOXA-143 (2) | blaOXA-58 + blaOXA-143 + blaGES-5 (1) | |
| blaIMP-1 + blaOXA-143 (1) | blaOXA-58 + blaOXA-143 (4) | |
| blaOXA-143 (2) | blaOXA-24/40 + blaOXA-143 (2) | |
| blaIMP-1 + blaOXA-143 (1) | ||
| blaIMP-7 + blaOXA-143 (1) | ||
| blaOXA-143 (6) | ||
| Aeromonas spp. (7) | blaKPC-2 (4) | blaKPC-2 (1) |
| blaGES-5 (1) | blaGES-16 (1) | |
| Citrobacter sp. (1) | blaKPC-2 + blaOXA-370 | NDb |
| Enterobacter spp. (68) | blaGES-5 + blaKPC-2 (1) | blaGES-16 + blaKPC-2 (2) |
| blaGES-16 + blaKPC-2 (1) | blaKPC-2 (52) | |
| blaKPC-2 (9) | ||
| blaKPC-26 (2) | ||
| blaGES-5 (1) | ||
| Klebsiella spp. (20) | blaKPC-2 (2) | blaKPC-2 (15) |
| blaKPC-26 (1) | ||
| blaGES-16 (1) | ||
| blaNDM-1 (1) | ||
| Kluyvera spp. (2) | blaKPC-2 | blaKPC-2 |
| Pseudomonas spp. (3) | blaVIM-2 | blaSPM-1 (1) |
| blaVIM-2 (1) | ||
| Serratia spp. (7) | NDb | blaKPC-2 (7) |
Suitable, ≤1,000 MPN/100 ml thermotolerant coliforms or ≤100 MPN/100 ml enterococci (10).
ND, not detected.
Klebsiella pneumoniae carbapenemase (KPC), GES-like carbapenemase, and NDM producers have been documented in recreational coastal waters impacted by sewage (7–9). Here, we observed that not only KPC, GES-like carbapenemase, and NDM but also IMP, VIM, SPM, and OXA carbapenemase-producing Enterobacteriaceae; Acinetobacter spp.; Aeromonas spp.; and Pseudomonas spp. can be recovered constantly from tourist coastal waters of Rio de Janeiro. The most frequent resistance determinant found in our study, blaKPC-2, is largely disseminated in hospital settings in Brazil and has been reported in hospital sewage and urban river water from Rio de Janeiro (2, 21). Curiously, in addition to the new blaKPC allele identified here (blaKPC-26), variants such as blaGES-16, blaIMP-16, blaOXA-370, and blaOXA-143 were so far reported only (or predominantly) in clinical isolates from Brazil (22–24). These findings reinforce that antimicrobial resistance determinants are exchanged between clinical and environmental settings.
Health implications of recreational exposure to these microorganisms remain unexplored. It is well documented that the bacterial species we studied here may incorporate into the human microbiota, and thus their dissemination throughout the community may happen extensively and silently. Given that carbapenemase producers found in coastal waters are known opportunistic pathogens, once the opportunity is given, colonized individuals may develop difficult-to-treat infections resulting in poor outcomes when inadequate therapy is implemented (25, 26). Because these opportunistic pathogens are found in water sources contaminated by sewage around the globe, this situation is probably not restricted to Rio de Janeiro and deserves attention from the scientific community and policy makers worldwide.
Accession number(s).
The nucleotide sequence of new variant blaKPC-26 was deposited in the GenBank database with accession number KX619622.
ACKNOWLEDGMENTS
We are grateful to Ana Cristina Gales, Laurent Poirel, and Ana Paula Carvalho Assef for providing the positive controls used in this study. We also thank Luiz Felipe Madeira for assistance in figure design.
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), which provided a research grant and productivity in research fellowship to R.C.P. (process numbers 482841/2013-3 and 311946/2016-0) and scholarships to R.P.P., L.F.M., and L.L.C.; by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), which provided a postdoctoral fellowship to E.H.C. and scholarships to L.R.L.B.; and by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), which provided a Young Investigator Grant to R.C.P. (process number E-26-201.555/2014).
The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.
R.C.P. had full access to all of the data in the study at all times and had final responsibility for the decision to submit for publication.
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