Skip to main content Accessibility help



  • Access


      • Send article to Kindle

        To send this article to your Kindle, first ensure is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

        Note you can select to send to either the or variations. ‘’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

        Find out more about the Kindle Personal Document Service.

        Different Escherichia coli B2-ST131 clades (B and C) producing extended-spectrum β-lactamases (ESBL) colonizing residents of Portuguese nursing homes
        Available formats

        Send article to Dropbox

        To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

        Different Escherichia coli B2-ST131 clades (B and C) producing extended-spectrum β-lactamases (ESBL) colonizing residents of Portuguese nursing homes
        Available formats

        Send article to Google Drive

        To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

        Different Escherichia coli B2-ST131 clades (B and C) producing extended-spectrum β-lactamases (ESBL) colonizing residents of Portuguese nursing homes
        Available formats
Export citation


ESBL-producing Enterobacteriaceae and particularly Escherichia coli ST131 isolates producing CTX-M enzymes are commonly found colonizing the intestine of nursing home (NH) residents, but ST131 subclonal structure has been scarcely explored in this vulnerable population. Our goal was to perform a pilot study to assess the faecal carriage rate and epidemiological features of ESBL- and/or carbapenemase-producing Enterobacteriaceae (ESBL-E and CPE, respectively) among NH residents. For this purpose, faecal samples from residents at 4 different NHs in the North of Portugal (representing 9·5% of the residents’ population, July 2014) were screened for ESBL-E and/or CPE by phenotypic and genotypic methods. Clonal structure and plasmid typing of ESBL-producing E. coli (ESBL-Ec) was performed by PCR and sequencing. Four ESBL-Ec isolates (2 CTX-M-15/2 CTX-M-14) were found in 20% of the samples, all belonging to the pandemic clonal lineage B2-ST131-O25b:H4. Two different clades were identified, the C2/H30-Rx-virotype C producing CTX-M-15 and an atypical B/H22-like-virotype D5 (producing CTX-M-14 and fluoroquinolone-resistant), firstly described in Portugal. This pilot study highlights the role of NH residents as a source of different ST131 clades, besides emphasizing the importance of E. coli B2-ST131 subtyping in different clinical settings, and understanding the transmission dynamics of the different variants.

Nursing home (NH) residents are known to be reservoirs of multidrug-resistant (MDR) bacteria, mainly due to their frequent hospitalizations, recurrent use of invasive medical devices and high antibiotic consumption [1]. Variable rates of intestinal colonization by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) (6–41%) have been reported in European countries, while carbapenemase-producing Enterobacteriaceae (CPE) have not yet been identified [25]. CTX-M-producing Escherichia coli Sequence Type (ST) 131 clone dominates by far the population of MDR Enterobacteriaceae colonizing the intestine of NH residents [3], but detailed analysis of ST131 subclonal structure has been scarce. In Portugal, ESBL-E (and particularly CTX-M-15-producing E. coli ST131 or Klebsiella pneumoniae ST15 clones) are endemic for several years in the clinical setting [68], whereas CPE (mainly KPC-3-producing K. pneumoniae) are quickly penetrating in our geographic region since the end of 2015, especially on susceptible populations [9]. The aim of this work was to perform a pilot study to assess the current faecal carriage rate of ESBL-E or CPE among NH residents in Portugal, and the clonal and subclonal structure of these isolates.

Fresh rectal swabs from 20 residents at four NHs located in the North of Portugal (5–6 km distance between them) were collected in July 2014 and analysed. Five residents per NH (ten females, ten males) were recovered, representing 9·5% of the total residents’ population. Eighty-five per cent of residents were ⩾65 years old (mean age of 75 years), 70% were previously hospitalized and all of them received antibiotic treatment during the 3 months preceding sampling (Supplementary Table S1). Samples were suspended in 2 ml of saline and screened for Enterobacteriaceae resistant to third-generation cephalosporins and/or carbapenems by seeding 0·2 ml of the suspension on CHROMagar™ Orientation plates supplemented with vancomycin (4 mg/l) plus ceftazidime (1 mg/l) or ertapenem (0·25 mg/l), respectively, and further incubation (37 °C/24 h) [10]. Presumptive Enterobacteriaceae isolates (oxidase negative, each different morphotype per plate) were selected for further studies. ESBLs and/or carbapenemases were identified by the DDST and Blue-Carba test, respectively, followed by polymerase chain reaction (PCR) and sequencing [10]. Susceptibility testing to non-β-lactam antibiotics was performed by the disk diffusion method ( and presumptive E. coli ESBL producers were identified by species-specific PCR [10]. The clonal structure of ESBL-producing E. coli was analysed by identification of E. coli phylogenetic groups and MLST ( [10]. Subclonal typing of B2-ST131 isolates was performed by PCR or PCR and sequencing of markers for ST131 serogroups (O25b:H4, O16:H5), clades (A, B, C1, C2) and fimH TR allele, and virulence genes (ibeA, iroN, sat, afa/draBC, papG allele II/III, cnf1, hlyA, cdtB, K1) [10, 11]. Plasmid analysis included replicon typing and subtyping (IncF plasmids) by PCR and sequencing (

Intestinal colonization by ESBL-E was detected in 4/20 (20%; 95% confidence interval (CI) 5·7–43·7) of the residents (Table 1), a colonization rate similar to that (24·5%) reported previously in our country in a larger sample from residents at NHs and long-term care facilities (LTCFs) [8]. These NH are managed by the same institution, share the nursing team and are served by the same hospital (H1). However, the asymmetry in the colonization rates observed (varying from 0% in NH1/NH2, 20% in NH3 and 60% in NH4) might be explained by the higher number of bedridden residents in NH3 and NH4 at sampling, which are at a higher risk of acquisition of MDR bacteria.

Table 1. Epidemiological data of ESBL-producing E. coli isolates identified in faecal samples from NH residents in Portugal

a F, Female, M, Male.

b PhG, E. coli phylogenetic group.

c ST, Sequence Type.

d IncF plasmids were identified using the FAB formula (FII, FIA, FIB) as proposed in

e Variability among isolates is shown in parenthesis.

f CIP, ciprofloxacin; NAL, nalidixic acid; STR, streptomycin; SUL, sulphonamides; TET, tetracycline; TMP, trimethoprim.

g fimH161, one SNP to fimH22.

The four NH residents positive for ESBL-E had recognized risk factors for ESBL-E carriage, such as previous antibiotic exposure and hospitalizations, but there was no significant statistic association between colonization and demographic (age, gender) or clinical (previous antibiotic treatment or hospitalization) data (Supplementary Table S1). Besides the low sample size, the absence of CPE is noteworthy but might not reflect the current situation since sampling occurred before the burden of CPE producers in clinical settings [9, 12].

All the ESBL-E were identified as E. coli producing CTX-M-15 (n = 2; two samples) or CTX-M-14 (n = 2; 2 samples) from different residents (Table 1). The species and the ESBL-types detected in our study are in line with the recent epidemiological trends in Portuguese hospitals [6], and with those observed in NHs from different European countries [3, 4, 8]. All ESBL-producing E. coli belonged to the pandemic B2-ST131-O25b:H4 clone and different clades thereof (C2/H30-Rx and B/H22-like). For both of them, the previous hospitalization of residents in the same hospital suggests nosocomial acquisition (Table 1). The C2/H30-Rx clade producing CTX-M-15 (n = 2) was identified in two residents from the same institution (NH4). It belonged to virotype C (sat), presented a MDR pattern and harboured only N and X4 plasmid replicons, instead of the typical IncF plasmids (Table 1) [13]. In fact, this clade corresponds to the most worldwide disseminated within E. coli B2-ST131 including in Portugal (Novais Â, unpublished results) [3]. Interestingly, B2-ST131-H30 virotypes A and B, previously associated with NH residents, were not detected in our sample [3]. Isolates from the less common clade B/H22-like (fimH161, differing in one SNP from fimH22) were identified in residents from NH3 and NH4, belonged to virotype D5 (ibeA, iroN, cnf1, hlyA), were MDR and produced CTX-M-14, and carried a higher diversity of plasmid replicons [I1, HI2, ColE, and an F2:A-:B1 virulence plasmid (resembling pAPEC-O2-ColV, GenBank accession number AY545598)] (Table 1). This clade (B/fimH22), firstly described in our country, is usually linked to community-acquired infections, but infrequently to fluoroquinolone resistance or ESBL production as reported in this study, which deserves further monitoring [3, 13]. This study, together with previous data, highlights circulation of different ST131 clades in diverse clinical and non-clinical settings in our country: (i) clade C2/H30-Rx in different Portuguese hospitals, NHs and LTCFs (this study, data not shown); (ii) clades C1-M27 and C1-nM27 in hospitals and healthy volunteers [10]; and (iii) an atypical clade B in NHs (this study).

In summary, this pilot study among NH residents in our country pointed-out the role of this setting as a source of different CTX-M-producing E. coli B2-ST131 clades (CTX-M-15-clade C2/H30-Rx and CTX-M-14-clade B/H22-like). Our data underscore the importance of B2-ST131 subtyping in different settings and further evaluation of transmission dynamics of the different subclones.


The supplementary material for this article can be found at


We are grateful to the residents, to the nurse José Feliciano Lopes, the director of healthcare services, in-charge for sample collection and the Management Board (Provedoria da Santa Casa da Misericórdia) of the nursing homes that participated in this study. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. CR and ÂN were supported by fellowships from FCT and Programa Operacional Capital Humano (POCH) (SFRH/BD/84341/2012 and SFRH/BPD/104927/2014, respectively).


None to declare.


1. Cassone, M, Mody, L. Colonization with multidrug-resistant organisms in nursing homes: scope, importance, and management. Current Geriatrics Reports 2015; 4: 8795.
2. Cochard, H, et al. Extended-spectrum β-lactamase-producing Enterobacteriaceae in French nursing homes: an association between high carriage rate among residents, environmental contamination, poor conformity with good hygiene practice, and putative resident-to-resident transmission. Infection Control and Hospital Epidemiology 2014; 35: 384389.
3. Nicolas-Chanoine, MH, Bertrand, X, Madec, JY. Escherichia coli ST131, an intriguing clonal group. Clinical Microbiology Reviews 2014; 27: 543574.
4. Willemsen, I, et al. Extensive dissemination of extended spectrum β-lactamase–producing Enterobacteriaceae in a Dutch nursing home. Infection Control and Hospital Epidemiology 2015; 36: 394400.
5. Saegeman, V, et al. Performance of different culture methods and of a commercial molecular assay for the detection of carbapenemase-producing Enterobacteriaceae in nursing homes and rehabilitation centers. European Journal of Clinical Microbiology and Infectious Diseases 2015; 34: 991997.
6. Rodrigues, C, et al. Increase of widespread A, B1 and D Escherichia coli clones producing a high diversity of CTX-M-types in a Portuguese hospital. Future Microbiology 2015; 10: 11251131.
7. Rodrigues, C, et al. Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized patients: a successful story of international clones (ST15, ST147, ST336) and epidemic plasmids (IncR, IncFIIK). International Journal of Medical Microbiology 2014; 304: 11001108.
8. Gonçalves, D, Cecílio, P, Ferreira, H. Nursing homes and long-term care facilities: reservoirs of CTX-M-15-producing Escherichia coli O25b-ST131 in Portugal. Journal of Global Antimicrobial Resistance 2016; 7: 6971.
9. Rodrigues, C, et al. KPC-3-producing Klebsiella pneumoniae in Portugal linked to previously circulating non-CG258 lineages and uncommon genetic platforms (Tn4401d-IncFIA and Tn4401d-IncN). Frontiers in Microbiology 2016; 7: 1000.
10. Rodrigues, C, et al. An update on faecal carriage of ESBL-producing Enterobacteriaceae by Portuguese healthy humans: detection of the H30 subclone of B2-ST131 Escherichia coli producing CTX-M-27. Journal of Antimicrobial Chemotherapy 2016; 71: 11201122.
11. Matsumura, Y, et al. Rapid identification of different Escherichia coli Sequence Type 131 clades. Antimicrobial Agents and Chemotherapy 2017; 61: e00179-17.
12. Manageiro, V, et al. Predominance of KPC-3 in a survey for carbapenemase-producing Enterobacteriaceae in Portugal. Antimicrobial Agents and Chemotherapy 2015; 59: 35883592.
13. Pitout, JDD, DeVinney, R. Escherichia coli ST131: a multidrug-resistant clone primed for global domination. F1000Research 2017; 6: 195.