Environmental microbiota represents a natural reservoir for dissemination

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1 AAC Accepts, published online ahead of print on August 0 Antimicrob. Agents Chemother. doi:./aac.001- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Environmental microbiota represents a natural reservoir for dissemination of clinically relevant metallo-β-lactamases Running title: Metallo-β-lactamases and environmental microbiota Claudia Scotta 1, Carlos Juan 1,,, Gabriel Cabot,, Antonio Oliver 1,,, Jorge Lalucat 1, Antonio Bennasar 1,, Sebastián Albertí 1,,*. Author s affiliation; 1. Área de Microbiología, Universidad de las Islas Baleares, Palma de Mallorca, Spain.. Instituto Universitario de Investigaciones en Ciencias de la Salud, Universidad de las Islas Baleares, Palma de Mallorca, Spain.. Servicio de Microbiología y Unidad de Investigación, Hospital Son Espases, Palma de Mallorca, Spain. * Corresponding author; Dr. S. Albertí Address: Edificio Científico-Técnico, CAMPUS-UIB. Crtra. Valldemosa, km.. Palma de Mallorca 01. Spain. Telephone: Fax: sebastian.alberti@uib.es. Downloaded from on October 1, 01 by guest - 1 -

2 A total of ten metallo-β-lactamase-producing isolates of six different species including Brevundimonas diminuta (), Rhizobium radiobacter (), Pseudomonas monteilii (1), Pseudomonas aeruginosa (), Ochrobactrum anthropi (1) and Enterobacter ludwigii (1) were detected in the sewage water of a hospital. The presence of bla VIM-1 associated to a Tn-class 1 integron structure was detected in all but one of the isolates (E. ludwigii, that produced VIM-), and in two of them (R. radiobacter), this structure was located in a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-β-lactamase genes. Downloaded from on October 1, 01 by guest - -

3 Metallo-β-lactamases (MBLs) have emerged worldwide as a major source of acquired broad spectrum β-lactam resistance. They virtually hydrolyze all classes of β-lactams (except monobactams), including carbapenems, which often represent the last option for the treatment of infections by multidrugresistant Gram-negative bacteria. There are two dominant types of transferable MBLs among the clinical isolates, IMP and VIM (1). Most of the IMP- and VIM-type MBL genes are present as gene cassettes inserted into integrons located in the chromosome or in plasmids. These integrons may be associated to transposon-like structures which may contribute to their variable location and spread (1). Interestingly, these MBL genes have been found almost exclusively in the hospital setting and the role of nonclinical habitats as a reservoir for bacteria that carry these acquired resistance determinants has been poorly investigated (, ). In this study, we evaluated the presence of MBL-producing bacteria in the sewage water of Son Llatzer Hospital (Mallorca, Spain) in order to obtain epidemiological data which could complement the results from a previous survey that investigated the incidence of MBL-producing Pseudomonas aeruginosa strains in this hospital (). For this purpose, duplicates of up to six ten-fold dilutions of 1 liter of sewage water collected 0 m downstream of the hospital wastewater discharge site were plated on three different media specially designed for the selection of Pseudomonads (Gould S1 Agar (1), King B Agar (), or Cetrimide Agar (Merck, Darmstadt, Germany)) containing 0 µg/ml of ceftazidime (Combinopharm, Madrid, Spain). A total of bacterial isolates, corresponding to different colony morphologies, were collected using this strategy and tested for the presence of Downloaded from on October 1, 01 by guest - -

4 MBL using the MBL E-test (biomerieux, Marcy l Etoile, France). Only sixteen isolates were positive in this test according to the manufacturer s instructions. Despite they exhibited different colony morphology, identification of these 1 MBL E-test positive isolates by 1S rrna gene amplification and sequencing as previously described (1), showed that they belonged to only eight different species including Brevundimonas diminuta, Rhizobium radiobacter, Pseudomonas monteilii, P. aeruginosa, Ochrobactrum anthropi, Enterobacter ludwigii, Acinetobacter johnsonii and Stenotrophomonas maltophilia (Table 1). Isolates belonging to the same species were not genetically related, as we demonstrated by ERIC-PCR () (data not shown). Furthermore, pulsed-field gel electrophoresis (PFGE) typing (), revealed that both P. aeruginosa isolates were different and not related with the MBL-producing P. aeruginosa clinical isolate PA-SL collected in a previous survey () (data not shown). Given that S. maltophilia produces an intrinsic MBL, we did not further investigated those isolates. In order to verify the presence of MBLs in the MBL E-test positive isolates we determined their ability to hydrolyze imipenem and whether this hydrolysis was EDTA sensitive (). The capacity to hydrolyze imipenem that exhibited all isolates was inhibited by EDTA in all cases, except for the A. johnsonii isolates which showed only weak hydrolysis and was not inhibited (Table 1). Moreover, the A. johnsonii isolates were found to produce the class D carbapenemase OXA- by PCR and sequencing, suggesting a false positive result of the MBL E-test. In contrast, PCR amplification using primers specific for bla VIM-1, bla VIM- and bla VIM-1 and conditions previously described (, ), revealed the presence Downloaded from on October 1, 01 by guest of VIM-type MBLs in all other isolates (Table 1). Interestingly, sequence - -

5 analysis of the resulting amplicons revealed the presence of bla VIM-1 (), in all isolates except E. ludwigii, which presented bla VIM- (Table 1). Moreover, the P. monteilii isolate was positive for both, bla VIM-1 and bla VIM-. Given that bla VIM-1 was predominant among the collected isolates, we focused on the characterization of the microorganisms harbouring this MBL gene. The susceptibility of the bla VIM-1 harbouring isolates and reference strains to a number of antibiotics was determined by E-test (biomerieux) following the manufacturer s instructions (Table ). All bla VIM-1 harbouring isolates had a similar pattern of multiresistance showing, in addition to the MBL mediated resistance to penicillins, cephalosporins, and carbapenems, resistance to gentamicin, tobramycin and amikacin with the exception of P. monteilii isolate that was susceptible to the aminoglycosides. Furthermore, most of them showed increased resistance to ciprofloxacin and trimethoprimsulfamethoxazole compared to the reference strain. All isolates were uniformly susceptible to colistin and minocycline, except B. diminuta and O. anthropi, which were resistant to colistin, and P. aeruginosa, which was resistant to minocycline. PCR amplification and sequencing, using the primers and conditions previously described (), evidenced that the integron harbouring bla VIM-1, was identical in all isolates and identical to the integron previously described in P. aeruginosa clinical isolate PA-SL (GenBank accession number; EF0.1) () (Figure 1). According to previously published data (), bla VIM-1 in PA-SL is located in a class 1 integron, where it is left-flanked by inti1 and right-flanked by aaca and qace 1. Furthermore, compared to bla VIM-1, bla VIM-1 shows higher efficiency for the hydrolysis of carbapenems but lower for ceftazidime and cefepime, due to Downloaded from on October 1, 01 by guest - -

6 two substitutions (LeuHis, ArgSer) within the active site centre (). Extended cloning and sequencing experiments in this work revealed that, in P. aeruginosa PA-SL, the integron harbouring bla VIM-1 was left-flanked by the resolvase and the transposase-encoding genes (tnpr and tnpa, respectively) of the Tn transposon (GenBank accession number; EU1) (Fig. 1) suggesting that the integron could be mobilized by the Tn machinery. Moreover, the integron contained four open reading frames including tnib (GenBank accession number; AJ0) (Fig. 1) which could represent a remnant of the original tni locus of the Tn0/In1 integron. To investigate whether the genes flanking the integron harbouring bla VIM-1 in PA-SL were also present in the environmental isolates, we performed a series of PCR amplifications using the set of primers represented in figure 1. As it is shown in figure 1, all B. diminuta isolates, one R. radiobacter isolate and one P. aeruginosa isolate showed positive PCR reaction with all the pairs of primers. Furthermore, all PCRs yielded products of the same size as those obtained with DNA from the clinical P. aeruginosa strain PA-SL. The other P. aeruginosa isolate and isolates of R. radiobacter and P. monteilii yielded PCR products with identical sizes to PA-SL on the right flank of the integron, while on the left flank they showed different results that ranged from positive reactions with PCR products larger than the expected sizes to absence of amplification. Finally, genomic DNA amplification of the O. anthropi isolate was only positive with one pair of primers located on the left side of the integron which yielded a product 00 bp larger to that observed in PA-SL. The presence of bla VIM -like gene in a transposon-integron structure in both, clinical and environmental microorganisms, has been reported for other MBL genes such as bla VIM- (), Downloaded from on October 1, 01 by guest - -

7 suggesting that the MBL integrons could be mobilized using the transposon machinery. To investigate whether the wide spread of bla VIM-1 among different environmental microorganisms was due to the location of this gene in a plasmid, genomic DNA of each of the environmental isolates harbouring bla VIM- 1 was digested with I-Ceu1, separated by PFGE and hybridized with bla VIM-1 and rrna probes as described previously (). In the R. radiobacter isolates the bla VIM-1 probe hybridized with bands which did not hybridized with the rdna, suggesting that in these isolates the bla VIM-1 gene is located in a plasmid, whereas matching of bla VIM-1 and rrna probes in all other isolates indicated chromosomal location (data not shown). Altogether our results suggest that environmental microbiota may represent an important reservoir of genetic determinants of antimicrobial resistance such as class 1 integron harbouring bla VIM-1, and that the sewage habitat can be considered a gathering place where many different species, including potential pathogens like P. aeruginosa, exist and those genetic determinants might be transferred among them. Downloaded from on October 1, 01 by guest - -

8 This work was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, through the Spanish Network for the Research in Infectious Diseases (grants REIPI C0/1 and RD0/ 000) and by the Govern de les Illes Balears (grant PROGECIC-C). Claudia Scotta was the recipient of a predoctoral fellowship from the Conselleria d Interior, Direcció General de Recerca, Desenvolupament Tecnològic i Innovació del Govern de les Illes Balears. Downloaded from on October 1, 01 by guest - -

9 REFERENCES Gould, D. W. 1. New selective media for enumeration and recovery of fluorescent Pseudomonads of various habitats. Appl. Bacterol. :-.. Gutiérrez, O., et al. 00. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Spanish hospitals. Antimicrob Agents Chemother. 1:-.. Héritier, C., L. Poirel, D. Aubert, and P. Nordmann. 00. Genetic and functional analysis of the chromosome-encoded carbapenem-hydrolyzing oxacillinase OXA-0 of Acinetobacter baumannii. Antimicrob Agents Chemother. :-.. Juan, C., et al. 00. Characterization of the new metallo-β-lactamase VIM-1 and its integron-borne gene from a Pseudomonas aeruginosa clinical isolate in Spain. Antimicrob. Agents. Chemother. :-.. Juan, C., et al. 0. Metallo-β-lactamase-producing Pseudomonas putida as a reservoir of multidrug resistance elementes than can be transferred to successful Pseudomonas aeruginosa clones. J. Antimicrob. Chemother. :-.. King, E. O. 1. The simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. : Lauretti, L., et al. 1. Cloning and characterization of bla VIM, a new integron-borne metallo-β-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother. :1-. Downloaded from on October 1, 01 by guest - -

10 Moyà, B., et al. 00. Β-lactam resistance response triggered by inactivation of a nonessential penicillin-binding protein. PloS Pathog. : e00.. Quinteira, S., H. Ferreira, and L. Peixe. 00. First isolation of bla VIM- in an environmental isolate of Pseudomonas pseudoalcaligenes. Antimicrob. Agents. Chemother. :-.. Quinteira, S., and L. Peixe. 00. Multiniche screening reveals the clinically relevant metallo-β-lactamase VIM- in Pseudomonas aeruginosa far from the hospital setting: and ongoing dispersion process? Appl. Environ. Microbiol. :-.. Versalovic, J., T. Koeuth, and J. R. Lupski.. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1: Walsh, R. T., M. A. Toleman, L. Poirel, and P. Nordmann. 00. Metallo-β-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 1: Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane.. 1S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 1:-0. Downloaded from on October 1, 01 by guest - -

11 Figure 1. Structure of the class 1 integron carrying bla VIM-1 and its flanking regions in P. aeruginosa clinical isolate PA-SL and the environmental isolates. Panel A shows the genes in the class 1 integron carrying bla VIM-1 (white arrows) and the genes flanking the integron (grey arrows) in PA-SL. The black arrows below the genes indicate the position of primers used for PCR amplifications while numbers on the brackets indicate the size of the PCR products obtained using the genomic DNA from P. aeruginosa clinical isolate PA-SL as template. Panel B shows the results of the PCR amplification of the genomic DNA from environmental isolates harbouring bla VIM-1 with primers shown in panel A. Positive PCR reactions with products identical in size to those obtained with the genomic DNA of PA-SL are indicated with, while negative PCR reactions are indicated with -. Asterisks indicate positive PCR reactions with amplicon s sizes (in brackets) different to those obtained with the genomic DNA of PA-SL. Downloaded from on October 1, 01 by guest - -

12 Table 1. Characteristics of the MBL E-test positive isolates. MIC (µg/ml) Organism (isolate) a IMP b IMPEDTA IMP hydrolytic activity c,d EDTA inhibition d,e Enzyme B. diminuta (1) > 1. ±. VIM-1 B. diminuta () 1.1 ± 0.1 VIM-1 B. diminuta () >. ±. VIM-1 R. radiobacter () > < 1 1. ±.0 VIM-1 R. radiobacter () > < ±.0 VIM-1 P. monteilii () > < 1.0 ±.0 VIM-1VIM- P. aeruginosa () > <1. ± 1.1 VIM-1 P. aeruginosa () >. ±. VIM-1 O. anthropi () 1. ±. VIM-1 E. ludwigii () > < 1. ± 1.1 VIM- A. johnsonii (). ± 0. - OXA- A. johnsonii (1) 1. ± 0. - OXA- A. johnsonii (1). ±.1 - OXA- S. maltophilia (1) > ND ND ND S. maltophilia (1) > ND ND ND S. maltophilia (1) > ND ND ND PA-SL (positive control) 1. ±. VIM-1 PA dacb (negative control) ND ND 1.± 0. - AmpC H a Previously described VIM-1-producing P. aeruginosa strain PASL () and an AmpC-hyperproducing mutant of PAO1 () were used as positive and negative controls, respectively. Downloaded from on October 1, 01 by guest b IMP, imipenem c Mean (nm/min/mg protein) ± SD of three independent experiments. d ND, Not determined e indicates an inhibition of the hydrolytic activity > 0% - 1 -

13 Table. MICs of environmental isolates harbouring bla VIM-1. MIC (µg/ml) a Organism (strain or isolate) CAZ FEP PIP TZP ATM IMP MEN GEN TOB AMK CIP CST SXT MIN B. diminuta (ATCC ) > 1 1 > B. diminuta (1) > > 1 > > > > 1 > 1 > 0.0 B. diminuta () > > > > > 0.0 B. diminuta () > > > > > > > 1 > > 0.0 R. radiobacter () > > > > 1 > > > > <0.0 > 1. R. radiobacter () > > > > 1 > > > > <0.0 > 1. P. monteilii (ATCC 00) P. monteilii () - > > 0. > 0. > P. aeruginosa (PAO1) P. aeruginosa (PA-SL) > > 1 > 1 > P. aeruginosa () > > 1 1 > P. aeruginosa () 1 1 > > 1 O. anthropi (CCUG ) > 1 > > > O. anthropi () > > > > > a CAZ, ceftazidime; FEP, cefepime; PIP, piperacillin; TZP, piperacillin-tazobactam; ATM, aztreonam; IMP, imipenem; MEM, meropenem; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin; CST, colistin; SXT, trimethoprim-sulfamethoxazole; MIN, minocycline Downloaded from on October 1, 01 by guest

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15 A tnpa tnpr intl vim1 aaca qace 1 sul1 orf tnib B 1. B. diminuta. B. diminuta. B. diminuta. R. radiobacter. R. radiobacter. P. monteilii. P. aeruginosa. P. aeruginosa. O. anthropi TnpAV1F TnpRintF1 IntFI QacE 1F SulFINR SulFINF TniBV1F 00 bp (00) * (1,00) * - (1,0) * 1,0 bp - * (1,0) - - 1,00 bp -,000 bp - 00 bp - Downloaded from on October 1, 01 by guest