Occurrence and spread of SHV extended-spectrum -lactamase-producing Klebsiella pneumoniae isolates in Curacao

Sir, In recent years, the growing incidence of extended-spectrum β-lactamases (ESBLs), in particular produced by Klebsiella pneumoniae isolates in nosocomial infections, has also become evident in Curacao (Netherlands Antilles). Because of the island’s location, the occurrence of ESBLs may be a consequence of local emergence or import from other countries.1 To identify a possible source for the increase in these multidrug-resistant isolates, 65 non-duplicate clinical isolates of ESBL-producing K. pneumoniae were investigated having been collected in the St Elisabeth Hospital from November 1999 to June 2002. Identification and susceptibility testing were carried out using an automated system (Vitek, bioMerieux Vitek Inc., Hazelwood, USA). When MICs indicated the potential presence of an ESBL, a doubledisc synergy test (DDST) was used to confirm ESBL production.2 Ribotyping using EcoRI was carried out with an automated riboprinter (Qualicon Europe Ltd., Warwick, UK) as described previously.3 Fourteen isolates collected during 2001 from an outbreak in the neonatal intensive care unit (NICU) were considered separately, because of the isolated character of this event. Twelve isolates were collected from November 1999 to December 2000 (group A). Thirteen isolates were collected outside the NICU, during 2001 (group B). Twenty-six isolates were collected in the first 6 months of 2002 (group C). There was a significant increase in the isolation of ESBL-producing K. pneumoniae isolates in Curacao, from less than one per month in 1999/2000 to approximately five per month in the first half of 2002, which was only partly explained by better screening methods. Twenty-two distinct ribotype patterns were identified (Figure 1) and were compared to an international database containing more than 1800 ribotype patterns that was constructed during the framework of the Genetic Epidemiology Network for Europe (GENE) project (www.ewi.med.uu.nl/gene). Ten of the 22 patterns, found in 42 Curacao isolates, were identical to patterns in the GENE database and most were found in different countries. Interestingly, ribotype 77 was the most represented type (26 isolates) in Curacao and was also the ribotype with the largest number of isolates (98 isolates) of the 10 matching patterns from the GENE database. This ribotype was mainly found in the Americas, but also in Europe. Other geographically widespread ribotypes, found both in the Americas and in Europe, were ribotypes 122 (44 isolates in the GENE database), 438 (38 isolates) and 38 (22 isolates). These results indicate that most Curacao klebsiellae in our study (42 out of 65 isolates) do not represent endemic strains but are exchanged between the island and other parts of the world, and also many of these imported strains have a wide geographical distribution. To identify the β-lactamase profiles in these K. pneumoniae isolates, isoelectric focusing (IEF) was carried out. Most of the non-NICU isolates demonstrated a variety of IEF patterns, and 38 of the 51 non-NICU (75%) isolates showed a pI ≥ 8.3 suggestive of a CTX-M-type or AmpC β-lactamase. To detect possible CTX-M β-lactamases, isolates were screened with three sets of primers.4 All isolates were negative. The presence of a DHA-type AmpC enzyme was confirmed by PCR amplification in eight isolates (12%).5 To assess whether blaSHV-type β-lactamases were present in our isolates, PCR amplification with the primers SHV-Nhe-F (5′-GAGCGAAAGATCCACTTCG-3′) and SHV-Nhe-R (5′-GTATCCCGCAGATAAATCA-3′) was carried out to detect a 525 bp blaSHV gene-specific fragment (GenBank accession no. AF124984[SHV1], bp 262–281 and bp 786–767). The majority of SHV-type ESBLs can be detected by restriction analysis of the amplicon by NheI.6 Sixty-three isolates showed an amplification product in the SHV-NheI-PCR. Fifty-four isolates showed two NheI-digestion products and were considered SHV-ESBL positive. The 11 isolates that yielded no digestion products had a positive DDST, but their ESBL phenotype was probably the result of another type of β-lactamase; these isolates were collected early in the study (group A). Sixty-two of 65 isolates showed transferable ampicillin resistance; in 24 isolates, this was associated with transfer of genes encoding SHV ESBLs. Almost all of the isolates from the NICU outbreak of 2001 yielded SHV transconjugants. However, for the non-NICU isolates, the conjugation rate for SHV was low (15%). This may be explained by poor mobilization of the SHV carrying plasmid or competition with other resistance plasmids. Our data indicate that there is an ongoing outbreak of ESBLproducing K. pneumoniae in Curacao. Most of the ESBL-producing isolates (83%) appear to carry plasmid-encoded SHV-type βlactamases and this plasmid is not easily transferable from the majority of isolates. Isoelectric focusing indicated that most of the isolates harbour a diversity of β-lactamase genes, with an AmpC-type present in 12%. There is a prevalent clone, whose ribotype pattern indicated that it is was probably imported from the Americas or Europe.