Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons, and sul genes in Portugal

Bacteria colonizing the human intestine have a relevant role in the spread of antimicrobial resistance. We investigated the faecal carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in healthy humans from Portugal and analyzed the distribution of sul genes and class 1 and 2 integrons. Faecal samples (n = 113) were recovered from healthy persons (North/Centre of Portugal, 2001–2004) and plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Isolates representing different morphotypes/plate and antibiotic susceptibility patterns (n = 201) were selected. Isolates resistant to sulfonamides and/or streptomycin, gentamicin, and trimethoprim were screened (PCR and sequencing) for sul genes (sul1, sul2, sul3) and class 1 and 2 integrons. Presence of ESBLs was inferred using the double disk synergy test (DDST) and further confirmed by PCR and sequencing. ESBL producers were selected for clonal analysis, plasmid characterization and conjugation assays by standard methods. ESBL-producing isolates were found in 1.8% (2/113) of samples, corresponding to Escherichia coli of phylogroups A (n = 1) and B1 (n = 1) carrying transferable blaCTX-M-14 and the new blaTEM-153, respectively. A 80kb IncK plasmid bearing blaCTX-M-14 was found, being highly related to that widely spread among CTX-M-14 producers of humans and animals from Portugal and other European countries. sul genes were found in 88% (22/25; sul2-60%, sul1-48%, sul3-4%) of the sulfonamide resistant isolates. Class 1 integrons were more frequently found than class 2 (7%, 14/201 vs. 3%, 6/201). Interestingly, gene cassette arrangements within these platforms were identical to those commonly observed among Enterobacteriaceae from Portuguese food-producing animals, although aadA13 is here firstly described in Morganella morganii. These results reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes including blaESBLs, and highly transferable genetic platforms as IncK epidemic plasmids.


INTRODUCTION
Antimicrobial resistance has become a global public health problem, compromising the treatment of several infectious diseases. Acquisition of integrons and beta-lactamase genes by Enterobacteriaceae is increasingly recognized, being associated with resistance to multiple antibiotics Coque et al., 2008;Bush, 2010;Pitout, 2010). The production of extended-spectrum beta-lactamases (ESBLs) constitutes one of the currently most spread and relevant antibiotic resistance mechanisms, compromising the use of several beta-lactams. Portugal is one of the European countries with higher rates of ESBL producers in the clinical setting, with a shift from TEM or SHV variants to CTX-M-types noticed since 2003 ECDC, 2011).

Colonization of healthy subjects with antibiotic resistant
Enterobacteriaceae could contribute to the amplification of resistant bacteria both at community and nosocomial settings (Rodríguez-Baño et al., 2008;Valverde et al., 2008). High rates of antibiotic resistant Escherichia coli fecal isolates of healthy humans have been reported in different countries (London et al., 1994;van de Mortel et al., 1998;Zhang et al., 1998;Sáenz et al., 2001;Briñas et al., 2002;Bruinsma et al., 2003;Nys et al., 2004;Bailey et al., 2010). In Portugal, spread of multidrug resistant bacteria, including ESBL producers, in hospitals, healthy food-producing animals, food products and aquatic settings has been described (Machado et al., , 2008(Machado et al., , 2009Mendonça et al., 2007). Nevertheless, their occurrence and diversity in healthy populations is unknown. In order to better understand the epidemiology of multidrug resistant Enterobacteriaceae in Portugal and the contribution of different settings to the burden of infections with antibiotic resistant bacteria, we investigated, during the initial spread period of CTX-M enzymes, the faecal carriage of ESBL-producing Enterobacteriaceae, class 1 and class 2 integrons, and sul genes in Portuguese healthy humans.

BACTERIAL ISOLATES
From January 2001 to February 2004, a total of 113 nonduplicate faecal samples were recovered from randomly selected healthy humans [53% females; age ranging from 5 to 59 (n = 99) and 60 to 76 (n = 14) years old] living in the North and Centre of Portugal. Samples were from persons without previous exposure to antibiotic therapy, hospitals, or long-term care facilities at least in the 3-month period before sampling. Rectal swabs were immersed in transport medium, faeces suspended in 1 mL of saline, and aliquots of 200 μL seeded in MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Presumptive Enterobacteriaceae (oxidase-negative facultative aerobic Gram negative rods) were selected for further studies. Enterobacteriaceae isolates recovered in the same time period from Portuguese hospitals (n = 4; 2003-2004) or marine coastal waters (n = 1; 2003) close to clandestine discharge points of water streams contaminated with faecal coliforms, and producing ESBL-types similar to the ones identified in this study, were also included for clonal investigation and/or plasmid relationships analysis (Machado et al., , 2009).

ESBL DETECTION AND ANTIMICROBIAL SUSCEPTIBILITY
Each different morphotype growing on MacConkey agar with ceftazidime or cefotaxime was screened for ESBL production by the double disk synergy test (DDST) (Jarlier et al., 1988), and susceptibility testing to non-beta-lactam antibiotics (aminoglycosides, quinolones, sulfonamides, trimethoprim, tetracyclines, chloramphenicol) was carried out in positive isolates using the standard disk diffusion method (CLSI, 2007). Morphotypes corresponding to non-ESBL producers recovered from MacConkey agar with and without antibiotics were tested to streptomycin, gentamicin, trimethoprim and sulfonamides (CLSI, 2007).
The transferability of ESBL genes was assessed by filter mating assays with E. coli BM21R (nalidixic acid-and rifampicin resistant, lactose fermentation positive and plasmid-free) (Machado et al., 2008), and ESBL-encoding plasmids were identified by PCR-based replicon typing and further hybridization (bla ESBL , rep probes), as previously described (Novais et al., 2010). Plasmid relationships were established by comparison of restriction fragment length polymorphism (RFLP) patterns obtained after digestion with EcoRI, PstI, and HpaI restriction enzymes (Valverde et al., 2009).

CHARACTERIZATION OF INTEGRONS AND sul GENES
Isolates representing different morphotypes and resistance patterns to streptomycin, gentamicin, trimethoprim and/or sulfonamides were selected for screening of class 1 and class 2 integrons by PCR (Table 1), as these phenotypes are commonly associated with these genetic structures . Class 1 and class 2 integrons were further characterized by RFLP-typing and sequencing, as described . Integrons were designated by roman numbers and a subindex indicates the class to which each integron belongs, as previously described . Isolates resistant to sulfonamides were also screened for the presence of sulfonamide resistance genes (sul1, sul2, sul3) by PCR ( Table 1).
The proportion of faecal carriage of ESBL-producing Enterobacteriaceae was 1.8% (2 of 113 samples), corresponding to samples recovered in 2003 from two females, aged 21 and 76 years, respectively.
Both ESBLs were successfully transferred by conjugation, and resistance to streptomycin was co-transferred with the bla CTX-M-14 gene. A clonal relationship was not established between CTX-M-14-producing E. coli recovered from healthy volunteers and from Portuguese hospitalized patients or marine coastal waters (Machado et al., , 2009). However, the plasmid containing bla CTX-M-14 , a 80kb IncK-bla CTX-M-14 plasmid, was similar to that of hospitalized patients from Portugal and other European countries (Valverde et al., 2009;Cottell et al., 2011; data not shown).

ANALYSIS OF INTEGRONS
Class 1 and/or class 2 integrons were detected in 9% (19/201) of the isolates, being class 1 integrons more frequently found than

DISCUSSION
The rate of faecal carriage of ESBL-producing Enterobacteriaceae found in Portuguese healthy persons was similar to that reported in Spain and other countries during the same time period (Valverde et al., 2004;Moubareck et al., 2005;Rodrigues et al., 2005). In Portugal, available data on faecal carriage of ESBLproducing Enterobacteriaceae by healthy humans is scarce and restricted to children (Guimarães et al., 2009). However, several works demonstrate a wide dissemination of ESBLs in Portuguese clinical settings, healthy food-producing animals, food products and aquatic environments since 2003, accompanied by a shift in the ESBL-types from TEM-or SHV-types towards CTX-M enzymes (Machado et al., 2004(Machado et al., , 2008(Machado et al., , 2009Mendonça et al., 2007;Peixe and Machado, personal communication). In face of this scenario, the presence of ESBL-producing Enterobacteriaceae in the commensal intestinal flora of Portuguese healthy humans was not surprising, being even predictable that future studies will report an increase in the faecal carriage of ESBL producers, as demonstrated in other works (Valverde et al., 2004;Pallecchi et al., 2007;Vinué et al., 2009;Woerther et al., 2010). The ESBL-producing E. coli clones identified belonged to A or B1 phylogenetic groups. These phylogroups have traditionally been considered "commensal" (Johnson and Stell, 2000), although they have been increasingly recovered from human infections, very often from blood cultures (Moreno et al., 2006;Cooke et al., 2010;Rodríguez-Baño et al., 2012;Novais and Peixe, unpublished data). The detection of CTX-M-14producing E. coli isolates from hospitalized patients and marine coastal waters clonally unrelated (phylogroup D) to the one from a Portuguese healthy person in the same time period, but sharing an identical IncK plasmid carrying bla CTX-M-14 also identified in Spain and other countries Valverde et al., 2009;Cottell et al., 2011), highlights the role of transferable plasmids in the global and local spread of antibiotic resistance among different reservoirs. The TEM-153 reported by the first time in this study seems to evolve from bla TEM-1c (Leflon-Guibout et al., 2000), a bla TEM-1 variant identified among animals and predominant among faecal isolates from healthy humans (Briñas et al., 2002). This enzyme is highly similar to TEM-52 (99% homology at aminoacid level, G238S, and V267G), an ESBL-type widely spread in piggeries, chicken meat, and hospitals in Portugal and Europe Machado et al., 2008;Peixe and Machado, personal communication).
Although limitations derived from the low number of ESBLproducing isolates included in our study, these data suggest that bacteria colonizing healthy persons constitute a reservoir of new or known ESBL genes that could further evolve at the nosocomial setting and/or be responsible for future epidemic situations. Despite the initial reports of CTX-M-15 producers in several Portuguese hospitals in  and the recent studies indicating a high prevalence of CTX-M-15 among the faecal flora of healthy individuals (reflecting its current dominance in the ESBL pandemic) (Moubareck et al., 2005;Geser et al., 2012;Lonchel et al., 2012), this enzyme was not identified during the period analyzed. A later penetration of CTX-M-15 in Portugal (as occurred in other South European countries)  might have resulted in a lower colonization pressure in these years, in opposite of CTX-M-14 that was already spread in non-nosocomial niches (Machado et al., 2009).
In agreement with previous studies from the same time period (Kang et al., 2005;Skurnik et al., 2005), integrons were found in a low percentage of isolates (9%). All class 1 integron types identified in this study have been extensively found in the hospital and community settings, including Portuguese food-producing animals, with the exception of the integron type XXIV 1 (containing the gene cassette aadA13 conferring resistance to streptomycin and spectinomycin) Vinué et al., 2008;Ben Sallem et al., 2012). The gene cassette aadA13 seems to be widespread among Portuguese E. coli isolates from pig faeces since 1998 (Machado et al., 2008). Its identification in different species of Enterobacteriaceae including firstly in Morganella morganii, highlights the highly transmissibility of certain genetic elements carrying antibiotic resistance in this area. Class 2 integrons contents resembled that of Tn1825 and the widely disseminated Tn7 (Biskri and Mazel, 2003;Machado et al., 2005).
The occurrence of sul genes was similar to that reported in other studies, with sul2 being more commonly found than sul1, and sul3 being scarcely observed (Enne et al., 2001;Grape et al., 2003;Infante et al., 2005;Hammerum et al., 2006;Trobos et al., 2008;Bailey et al., 2010). These high rates of sul genes are identical to those detected among food-producing animals which are highly exposed to sulfonamides, and hence an involvement of the food chain cannot be discarded (Perreten and Boerlin, 2003;Pena et al., 2004;Antunes et al., 2005;Hammerum et al., 2006). The observation of sul3 in one isolate harboring intI1 but lacking sul1 suggests a replacement of sul1 by sul3, as previously reported in similar structures of Salmonella and E. coli isolates (Antunes et al., 2007;Curião et al., 2011). Although sul genes were not observed in some sulfonamide resistant isolates, we could not discard the presence of other resistance mechanisms, as mutations at the chromosomal gene (folP) for dihydropteroate synthase (DHPS) (Sköld, 2001) or acquisition of unknown genes.
Prevalence data on intestinal carriage of relevant antibiotic resistance genes and/or structures promoting gene expression in different time periods are important to identify sources and hotspots of antibiotic resistance with relevance for the human health. In summary, data from this retrospective study reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes (bla  or bla TEM153 ; sul)/genetic platforms (integrons and IncK plasmids). These findings impose future extensive follow-up evaluations in order to understand the trends of the antibiotic resistance genes epidemiology in the community and clinical settings over the time, and eventually anticipate the detection of microorganisms with the potential to cause pandemics in the future.

ACKNOWLEDGMENTS
We are very grateful to all persons who took part on this investigation, either as sample providers or recruiters.

FUNDING
The present work was supported by Fundação para a Ciência e Tecnologia, which belongs to the Ministry of Science, Technology and Innovation of Portugal (through grant no. PEst-C/EQB/LA0006/2011). Work in TMC lab is supported by grants in the EU (BIOHYPO 2008-227258). Elisabete Machado was supported by a fellowship from Fundação para a Ciência e a Tecnologia de Portugal (SFRH/BD/11304/2002).