Infections with extended spectrum β-lactamase (ESBL)-producing bacteria are a major public health threat worldwide and are associated with significant morbidity, mortality and increased healthcare costs . β-lactamases of the CTX-M group first emerged in the late 1980s and early 1990s and are now the most prevalent ESBL type reported worldwide . Their widespread dissemination can be attributed to their association with specific epidemic plasmids, e.g. IncFII; and specific clones, notably Escherichia coli O25b:H4-ST131, Klebsiella pneumoniae ST258, and K. pneumoniae ST11 . Community-acquired CTX-M-producing E. coli urinary tract infection frequently dominate reports; however, increasingly outbreaks associated with multidrug-resistant ESBL K. pneumoniae are reported [2, 3]. Most recent available data from the European Antimicrobial Surveillance Network indicate that across Europe the percentages of invasive K. pneumoniae resistant to third-generation cephalosporins range from 0% to 70·1%, with ESBL producers accounting for between 85% and 100% of all strains reported . Further, increasing combined resistance to fluoroquinolones, aminoglycosides and third-generation cephalosporins was also widespread with a reported mean percentage of 20·9% in 2013 . The most recent data for Ireland for 2014 indicate that 11·3% of invasive K. pneumoniae were ESBL producers, 17·3% were resistant to ciprofloxacin, 13·2% were resistant to aminoglycosides, and 13·7% were resistant to all three .
In late 2012 an outbreak of CTX-M ESBL-producing K. pneumoniae was identified at a neonatal intensive care unit (NICU) in Ireland , and two infants were diagnosed with bloodstream infection with this organism. All infants in the NICU during the outbreak period were screened by rectal swab for carriage of ESBL K. pneumoniae and 22 of 78 infants proved positive. No new cases of infection or carriage were detected after 31 October 2012 and no new rectal swabs were positive after 2 January 2013 indicating that transmission had been interrupted and no environmental reservoir was identified. All isolates were ciprofloxacin non-susceptible and gentamicin resistant in addition to ESBL (CipGeESBL). Prompted by the recognition that K. pneumoniae with this phenotype had been observed in other hospitals an investigation was undertaken to determine the extent of dissemination of such strains throughout Ireland.
Retrospective analysis of data stored by the Antimicrobial Resistance and Microbial Ecology (ARME) group identified 75 isolates of K. pneumoniae from eight hospitals throughout Ireland with the CipGeESBL phenotype since January 2011. In December 2012, hospital laboratories throughout the country were alerted to this concern and invited to submit K. pneumoniae isolates from all specimen types with this phenotype. Forty-four isolates were received from 11 hospital laboratories between December 2012 and July 2013 and the total collection comprised 137 isolates from 17 hospitals (including 18 isolates from the original NICU outbreak and four isolates from nursing home residents) between January 2011 and July 2013.
ESBL production was determined in all isolates by the combination disk method using cefpodoxime (30 g), and cefpodoxime plus clavulanic acid (10 μg/1 μg). All isolates were screened for susceptibility to the following antimicrobial agents by disk diffusion in accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria where available: ampicillin (10 μg); cefpodoxime (10 μg), cefotaxime (5 μg), ceftazidime (10 μg), cefoxitin (30 μg), amoxicillin-clavulanate (20 μg/10 μg), piperacillin-tazobactam (30 μg/6 μg), ertapenem (10 μg), meropenem (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), kanamycin (30 μg), streptomycin (10 μg), chloramphenicol (30 μg), sulphonamides (250 μg), tetracycline (30 μg), trimethoprim (5 μg), and minocycline (30 μg) . Clinical Laboratory Standard Institute (CLSI) interpretive criteria was applied to antimicrobial agents for which EUCAST interpretive criteria were not available.
Confirmed ESBL-producing strains were screened for bla
OXA, and bla
CTX-M by multiplex polymerase chain reaction (PCR) assays using primers and protocols as described previously [8, 9], and carbapenem-resistant isolates [n = 22] were screened for bla
IMP and bla
OXA-48 by real time PCR . Pulsed-field gel electrophoresis (PFGE) was performed on all isolates according to the PulseNet protocol with XbaI and DNA banding patterns were analysed using the Dice coefficient with clustering by the unweighted pair-group method with arithmetic averaging (UPGMA) . One or more isolates representative of major PFGE clusters were subjected to multilocus sequence typing (MLST) as described previously [12, 13]. Allele and profile were determined by comparison to data from the K. pneumoniae MLST database (www.pasteur.fr/mlst).
All isolates were confirmed as ESBL producers and harboured bla
SHV and a bla
CTX-M gene (bla
CTX-M-group-1, n = 126; bla
CTX-M-group-25, n = 5), bla
CTX-M-group-2 (n = 5), and bla
CTX-M-group-9 (n = 1). Four isolates received from hospital 11 (see Table 1 for hospital numbers) (n = 2), hospital 9 (n = 1) and hospital 10 (n = 1) were positive for bla
KPC-2 and one isolate from hospital 9 harboured bla
NDM and bla
OXA-48 (Table 1). All isolates were resistant to ampicillin, amoxicillin-clavulanate, cefpodoxime, ciprofloxacin, gentamicin and nalidixic acid, and at least ⩾2 other antimicrobial agents giving 66 individual antibiograms in the 137 isolates. One isolate was resistant to all antimicrobial agents tested; 15 isolates were resistant to ertapenem, seven of which were also resistant to meropenem.
Table 1. Correlation of pulsed-field profile with location, date of isolation, β-lactamase, and sequence types of multidrug-resistant Klebsiella pneumoniae
PFGE identified 111 different profiles which fell into 16 major clusters (A–P) in 112 isolates based on a similarity of ⩾85% (Table 1, Fig. 1) with each cluster comprising 2–43 isolates. The largest cluster (A, n = 43) accounted for 31% of all isolates and contained isolates from three hospitals between February 2011 and October 2013, including the 18 isolates from the initial NICU outbreak (Table 1, Fig. 1).
Fig. 1. Dendogram showing relatedness of representative isolates from clusters and individual profiles identified by pulsed-field gel electrophoresis (PFGE) and correlation with multilocus sequence typing (MLST) analysis
Seventeen sequence types (STs) were identified in 36 representatives of distinguishable pulsed-field patterns: ST1236 (n = 8), ST15 (n = 8), ST258 (n = 3), ST23 (n = 2), ST48 (n = 2) and ST307 (n = 2) (Fig. 1). Eight isolates from two hospitals representing PFGE clusters A, H, I and J and one unique isolate were designated a novel MLST type, ST1236, a single locus variant of ST48. All isolates of ST1236 were ⩾73% similar by PFGE analysis. Eight isolates received from three hospitals of PFGE cluster O and individual DNA profiles with ⩾73% similarity were assigned to ST15. Three isolates from individual hospitals were of ST258, and harboured bla
CTX-M-25 and bla
SHV and the fourth K. pneumoniae carbapenemase (KPC)-producing isolate fell in PFGE cluster F and by extrapolation to ST258. The single bla
OXA-48 isolate was assigned to ST14.
The dissemination of multidrug-resistant K. pneumoniae represents a very significant threat to patients' safety and public health with several reports of outbreaks in hospitals particularly in NICUs [14, 15]. Data generated in this study suggests that two major clonal groups (CG) of multidrug-resistant K. pneumoniae, ST1236/ST48 (CG43) and ST15/ST14 (CG15) have been circulating in Ireland since at least January 2011. By extrapolation from PFGE analysis, 57 isolates from five hospitals and a nursing home fell into CG43, and 26 isolates from three hospitals into CG15, both groups together accounting for 61% of the entire collection. Since the establishment of the MLST scheme for K. pneumoniae in 2005 , the database now comprises 1874 STs, several of which have been described as successful pandemic clones. Attempts have been made by a number of workers to group individual STs into clonal groups and clonal complexes although it is recognized that this is challenging in K. pneumoniae due to the high rate of recombination between STs [2, 12, 16, 17]. ST1236 was first defined in this study and is a single locus variant of ST48, a member of CG43  which has been disseminated worldwide  along with ST15 (CG15) . The single ST14 isolate harbouring bla
NDM and bla
OXA-1, and bla
CTX-M-group1 is a single locus variant of ST15, and has been widely reported in association with CTX-M, and to a lesser extent carbapenemases including NDM-1, OXA-181 (a variant of OXA-48) . The three KPC-producing isolates of ST258 are representative of a major pandemic clone comprising 96 STs .
This study therefore demonstrates that although there is considerable diversity among the CipGeESBL K. pneumoniae in Irish hospitals, a high proportion (61%) of the isolates were accounted for by two clonal groups, and in at least one case spread had extended into nursing home residents in the community.
Active surveillance to enhance knowledge of the distribution of these STs/clonal groups among ESBL-producing K. pneumoniae is required to inform the development and implementation of appropriate infection control and prevention procedures. Schwaber et al. recently described containment of a nationwide outbreak of KPC-producing K. pneumoniae in Israel by stringent application and monitoring of infection control procedures  but, nevertheless, they acknowledged that the success of such an approach is highly dependent on the commitment of leaders in health policy planning to act on the data produced by studies such as described here.
We thank participating hospital laboratories for provision of isolates, Stephen Murchan, and Robert Cunney, the Health Protection Surveillance Centre and EARS-Net for provision of epidemiological data, and the team of curators of the Institut Pasteur MLST system for importing novel alleles, profiles and/or isolates at http://
This work was partially financed by the grant SPUB MIKROBANK from the Polish Ministry of Science and Higher Education
DECLARATION OF INTEREST
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