Introduction

AmpC β-lactamases (AmpC) are clinically relevant cephalosporinases encoded on the chromosome of many Enterobacteriaceae. Some of them are also described on plasmids, usually in species that do not naturally produce AmpC, such as Klebsiella spp., Salmonella enterica or Proteus mirabilis.

Plasmid AmpC (pAmpC) derive from chromosomal ampC genes of the families Enterobacteriaceae and Aeromonadaceae. First characterised in 1988, pAmpC described to date can be grouped into seven families: ACC, CMY, DHA, FOX, MIR, ACT and MOX [1]. These enzymes have been identified throughout the world, with CMY-2 being the most prevalent. A previous study performed in Barcelona (Spain) showed a significant increase of pAmpC in Enterobacteriaceae from 0.06 % in 1999 to 1.3 % in 2007 [2], in agreement with other authors (0.13 % to 3 %) [3–6].

Classes A, B and D of acquired carbapenemases are also increasing in Enterobacteriaceae. Often involved in nosocomial outbreaks [7–12], these emerging enzymes are worrisome because their broad activity profiles encompass all β-lactam antibiotics. Additionally, carbapenemases are difficult to detect using phenotypical methods because their resistance level to carbapenems is frequently below the established clinical breakpoints [13, 14].

The prevalence of pAmpC and carbapenemases could be underestimated because of the lack of consensus on the methodology to detect them [15]. This study aimed to determine the prevalence of these enzymes in Enterobacteriaceae isolates from clinical samples collected in Spain over a six-month period in 2009.

Materials and methods

Study design, bacterial strains and susceptibility tests

A prospective multi-centre study was undertaken in Spain to collect pAmpC- or carbapenemase-producing Enterobacteriaceae isolates from clinical samples from February to July 2009. Thirty-five Spanish hospitals participated, six of which acted as co-ordinating centres. These hospitals were distributed throughout 14 of the 18 Autonomous Communities in Spain.

Only one isolate per patient was considered for analysis. Isolates were identified in each hospital, using standard methods. Disc-diffusion and microdilution susceptibility tests were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines [16]. As some pAmpC do not confer resistance to cefoxitin, each hospital collected all the strains showing resistance or intermediate susceptibility to both co-amoxiclav (<18 mm; >8/4 mg/L) and any of the following: cefotaxime (<23 mm; >8 mg/L), ceftazidime (<18 mm; >8 mg/L) or aztreonam (<21 mm; >8 mg/L).

All extended-spectrum β-lactamase (ESBL)-producing isolates were excluded, except those that were resistant to or had intermediate susceptibility to cefoxitin (<15 mm; >8 mg/L) or co-amoxiclav. ESBL production was studied using the CLSI confirmatory test [16], the double-disc synergy test [14] and/or Etest ESBL (AB Biodisk, Solna, Sweden). Enterobacteriaceae with inducible chromosomal AmpC β-lactamase were also excluded from this part of the study.

Isolates suspected of carrying a carbapenemase were first selected because they showed a minimum inhibitory concentration (MIC) to imipenem >1 mg/L. The modified Hodge test [17] and Etest MBL IP/IPI (AB bioMérieux, Solna, Sweden) were used to confirm carbapenemase activity.

Susceptibility to aminoglycosides, quinolones or co-trimoxazole was determined by broth microdilution (MicroScan, Siemens Healthcare Diagnostics, Deerfield, IL, USA).

Characterisation of pAmpC and carbapenemases

The detection and characterisation of their genes were performed using the specific primers listed in Table S1 (supplementary data) or as previously described [1, 7, 18–22]. All amplicons obtained were purified and sequenced as previously described [7].

PFGE

For clonal studies, a group of strains were selected according to the following criteria: (i) all carbapenemase-producing isolates, (ii) all pAmpC-producing isolates from the co-ordinating centres and (iii) two pAmpC-producing isolates per species and enzyme (one at the start and one at the end of the study period) from the remaining participant hospitals. The genetic relationship among isolates of the same species was determined by pulsed field gel electrophoresis (PFGE) using a CHEF-DRII device (Bio-Rad, Hercules, CA, USA) after total chromosomal DNA digestion with XbaI (Escherichia coli, Klebsiella pneumoniae, K. oxytoca, Enterobacter cloacae and Citrobacter koseri) [23] or NotI (P. mirabilis) [24]. DNA relatedness was calculated based on the criteria of Tenover et al. [25].

Nucleotide sequence accession numbers

The new β-lactamase gene sequences were submitted to GenBank under accession numbers HM544039 (bla CMY-54), HM544040 (bla CMY-55), HQ322613 (bla CMY-56), HQ285243 (bla CMY-57), HQ267531 (bla CMY-96), HQ322612 (bla DHA-6), HQ456945 (bla DHA-7), HM565917 (bla FOX-8) and HQ263342 (bla IMP-28).

Results and discussion

Our study showed that the prevalence of pAmpC and carbapenemases in Spain in 2009 was 0.64 % and 0.04 %, respectively. These data were obtained from 100,132 isolates of Enterobacteriaceae, of which 1,654 showed a susceptibility phenotype compatible with the production of pAmpC or carbapenemases. These results were similar to the prevalences found around the world [2, 4–6, 26].

Genes coding for pAmpC or carbapenemase were detected in 674 isolates (Table 1). Using phenotypic methods, we found that those isolates with negative polymerase chain reaction (PCR) results were ESBL-producers, serine class A-producers (Klebsiella spp.) or hyperproducers of chromosomal AmpC (E. coli). However, non-enzymatic resistance mechanisms such as altered permeability may have also contributed to resistance in these isolates [14].

Table 1 Prevalence of pAmpC and carbapenemases in Enterobacteriaceae isolated in Spain in 2009
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The highest prevalence of pAmpC in Spain was found in the Autonomous Community of Catalonia (0.92 %), followed by Asturias (0.85 %). The lowest prevalence was observed in the Balearic Islands (0.35 %). No cases of pAmpC were detected in Castilla y León (Figure S1, supplementary data). This geographical variability was probably due to both the number of centres and the different kind of healthcare institutions included per autonomous community.

The most prevalent pAmpC enzymes found were CMY (78.3 %), followed by DHA (19.5 %), ACC (1.6 %) and FOX (0.6 %) (Table 2). CMY-2 remained the most widely distributed, especially in E. coli (87.1 %) and P. mirabilis (88.8 %) species, whereas DHA-1 enzyme was the most predominant in Klebsiella spp. isolates. Remarkably, both these enzymes were also found in other Enterobacteriaceae species, such as C. koseri, P. penneri and S. enterica (Table 2, Figure S1, supplementary data). FOX-type enzymes were found exclusively in E. coli. Nucleotide sequence analysis of all bla genes identified eight new pAmpC (bla CMY-54, bla CMY-55, bla CMY-56, bla CMY-57, bla CMY-96, bla DHA-6, bla DHA-7 and bla FOX-8).

Table 2 Prevalence of pAmpC and carbapenemases in the 674 Enterobacteriaceae strains studied
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Table 3 shows the correlation between resistance to non-β-lactam antibiotics and the most frequent species/pAmpC enzymes. The resistance of pAmpC-producers to non-β-lactam antibiotics was high: 70.2 % to ciprofloxacin, 65.9 % to co-trimoxazole, 44.8 % to tobramycin, 35.8 % to gentamicin and 7.4 % to amikacin. Co-resistance to ciprofloxacin, co-trimoxazole, gentamicin and tobramycin was detected in 23.4 % of the isolates. Regarding resistance to non-β-lactams, we did not find any statistically significant differences between CMY-2- and DHA-1-producing isolates.

Table 3 Resistance to non-β-lactam antibiotics in the most frequent species/pAmpC associations
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VIM-1, VIM-2 and IMP-1 are the most frequent carbapenemases reported in Enterobacteriaceae around the world [27]. In Spain, the first VIM-1 enzyme was described in 2003 in K. pneumoniae and E. coli [7, 28]. Since then, several outbreaks involving K. pneumoniae and E. cloacae strains have been published [7, 28, 29]. In the present study, we found that the VIM-1 enzyme was the most frequent carbapenemase. We also detected IMP-22-producers and the new IMP-28-producing strain that was recently published [30]. As Gaibani et al. [31], we did not find any KPC, NDM or OXA carbepenemases. Our study, therefore, preceded the emergence of carbapenemases such as OXA-48 and NDM-1 that were recently described in Spain [32, 33]. The resistance of carbapenemase-producers to non-β-lactam antibiotics was high and similar to the pAmpC-producers: 60 % to ciprofloxacin, 80 % to co-trimoxazole, 33 % to tobramycin, 27.5 % to gentamicin and 7.5 % to amikacin. Co-resistance to ciprofloxacin, co-trimoxazole, gentamicin and tobramycin was detected in 17.5 % of the isolates.

A total of 324 non-duplicate isolates were selected to determine their molecular typing. PFGE analysis showed a great genetic diversity: 172 different PFGE patterns in 179 E. coli isolates, 51 in 67 K. pneumoniae isolates, 45 in 48 P. mirabilis isolates, 10 in 15 K. oxytoca isolates, 11 in 11 E. cloacae isolates and 3 in 3 C. koseri isolates. The PFGE results revealed four well-defined clusters: (i) all nine IMP-22-producing K. pneumoniae isolates from Hospital Puerta del Mar (Cadiz), (ii) five VIM-1-producing K. oxytoca from Hospital Ramón y Cajal (Madrid), (iii) three CMY-2-producing E. coli from Hospital Virgen del Rocío (Seville) and (iv) three CMY-2-producing P. mirabilis from Hospital Sant Pau (Barcelona). We also detected 15 pairs of identical isolates: seven pairs of CMY-2-producing isolates (four of E. coli and three of K. pneumoniae), three of VIM-1-producing isolates (two K. pneumoniae and one K. oxytoca), two of DHA-1-producing K. pneumoniae, one of AAC-1-producing K. pneumoniae, one of FOX-8-producing E. coli and one of DHA-7- and VIM-1-producing E. cloacae. All but one of these strain pairs consisted of isolates from the same hospital. The highest number of strain pairs was detected in Hospital Vall d’Hebron (Barcelona) and corresponded to three CMY-2-producing isolates (two of E. coli and one of K. pneumoniae) and one pair of E. cloacae isolates co-producing DHA-7 and VIM-1. Finally, one pair of VIM-1-producing K. oxytoca strains comprised of isolates from Hospital Vall d’Hebron and from Laboratori de Referència de Catalunya (both in Barcelona), but no epidemiologic relationship between the patients carrying these isolates could be demonstrated.

pAmpC-producing strains cause nosocomial, healthcare-associated and community infections mainly in predisposed patients. The clinical data of the patients included in our study have already been published, showing that invasive infections were associated with high mortality, which might be partly related to inappropriate empirical therapy [22].

In spite of the predominance of CMY-2- and DHA-1-producing E. coli, K. pneumoniae and P. mirabilis, we found a great genetic diversity among these isolates, higher in E. coli and P. mirabilis than in K. pneumoniae. This finding supports the theory that the spread of these antibiotic resistance mechanisms is mainly due to the dissemination of mobile genetic elements among a polyclonal population. Previous studies have demonstrated the polyclonal dissemination of CMY-2-producing E. coli isolates [23, 34, 35]. However, nosocomial outbreaks due to DHA-1- or ACC-1-producing K. pneumoniae or CMY-2-producing P. mirabilis have been only sporadically described [36, 37]. In this study, we describe two small nosocomial outbreaks due to CMY-2-producing E. coli and P. mirabilis, respectively.

MBL-producing clinical Enterobacteriaceae isolates have been reported all over the world [7, 38, 39]. In this study, we describe two nosocomial outbreaks in two hospitals in Spain, one due to VIM-1-producing K. oxytoca in Hospital del Mar (Barcelona) and the other due to IMP-22-producing K. pneumoniae in Hospital Puerta del Mar (Cadiz). Conejo et al. [9] described a nosocomial outbreak of IMP-8-producing K. oxytoca strain in the same period and in the same area. In contrast to the wide spread of the KPC-producing ST258 K. pneumoniae strain [10], the dissemination of VIM and IMP carbapenemases seems to be associated to both clonal intra-hospital spread of local strains [7, 8, 31] and to non-clonal dissemination [7, 29, 38].

The detection and surveillance of pAmpC- and carbapenemase-producing strains is needed in order to implement appropriate therapeutic options and appropriate infection control measures [13]. However, the lack of standardised phenotypic methods has become evident in this study, as only 63 % of all evaluated isolates (n = 1,654) were pAmpC- or carbapenemase-producers.

In conclusion, the prevalence of pAmpC and carbapenemases found is not negligible. The CMY-type enzymes were the predominant pAmpC found, whereas the VIM or IMP enzymes were the most frequent carbapenemases. A great genetic diversity among pAmpC-producing strains and a closer clonal relationship between carbapenemase-producing strains was found.