Abstract
The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008–2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify blaESBL (blaSHV, blaTEM, and blaCTX-M genes of groups 1, 2, 9, and 8/25) and blaKPC genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the blaESBL and blaKPC genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one blaESBL gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of blaTEM (n = 5), blaSHV (n = 11), blaCTX-M (n = 19), and blaKPC (n = 3) were detected. A new blaSHV variant, blaSHV-129, and a new blaKPC variant, blaKPC-11, were also identified. The most common bla genes found in this study were blaCTX-M-15, blaCTX-M-14, and blaSHV-12. Using nucleic acid microarrays, we obtained a “molecular snapshot” of blaESBL genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants blaSHV-129 and blaKPC-11.
INTRODUCTION
Resistance to extended-spectrum penicillins and cephalosporins among Gram-negative pathogens such as the Enterobacteriaceae is a serious and growing health care threat (31). Commonly, extended-spectrum β-lactamases (ESBLs) are responsible for this phenotype and are increasingly being reported among these microorganisms (26). As these multidrug-resistant ESBL-producing isolates are now often treated with carbapenems, carbapenemases, such as KPC, are also an emerging problem.
Single- and multiple-amino-acid substitutions around the active site in the parental narrow-spectrum TEM- and SHV-type enzymes (i.e., TEM-1 and -2 and SHV-1 and -11) are the basis of ESBL-mediated resistance. To date, more than 190 TEM-type and 140 SHV-type β-lactamases have been identified worldwide (www.lahey.org/Studies). During the last 10 years, other ESBL types have gained importance, especially the CTX-M enzymes. The CTX-M family is clustered in five subgroups, designated CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 (5). CTX-M enzymes are currently the most prevalent β-lactamases found in Escherichia coli clinical isolates worldwide (6).
The KPC-type β-lactamases, which confer resistance to extended-spectrum cephalosporins, β-lactam–β-lactamase inhibitor combinations, and carbapenems, present one of the most significant challenges to date (23, 25). Eleven variants have been reported on the Lahey Clinic website thus far. KPC-2 and KPC-3 are widespread and are the most frequently identified carbapenemases in Enterobacteriaceae. KPC-producing bacteria were identified first in the United States and subsequently on almost every continent (2, 9, 22, 23, 29, 30, 39). Detection of these enzymes remains difficult because the MICs of carbapenems for some blaKPC-producing isolates at times remain in the susceptible range, especially when automated systems are used. Furthermore, the presence of multiple β-lactamases often renders isolates resistant to β-lactam–β-lactamase inhibitor combinations, and the presence of AmpC enzymes may produce a negative confirmatory test for ESBL production, even if a blaKPC gene is present (15).
As a result of widespread dissemination of ESBLs and carbapenemases, numerous detection strategies have been developed for identification of ESBL- and KPC-producing microorganisms. Many researchers have found that standard phenotypic tests may fail, yielding unacceptable numbers of false-negative results (12, 17, 33). The lowering of clinical breakpoints has improved the sensitivity of phenotypic tests; nevertheless, it is prudent to remain cautious, since some isolates that give test results in the susceptible range may result in to clinical failure when part of an infection (11, 34). PCR followed by DNA sequencing (PCR-sequencing) is still the most widely used molecular technique for characterization of ESBLs; however, these methods remain time-consuming. Pyrosequencing and microarray technologies are promising genotyping systems which overcome these constraints (13, 14).
Few large-scale studies that examine the global distribution of ESBLs and carbapenemases present in clinical isolates are reported in the literature (27). These analyses provide a unique opportunity for molecular epidemiologists and clinicians to assess the potential impact of highly resistant Gram-negative pathogens on treatment and infection control strategies. The objective of this study was to characterize a major collection of 1,093 Gram-negative organisms with an ESBL phenotype from geographically diverse locations (Africa, Asia, Europe, Latin America, the Middle East, North America, and the South Pacific). All strains were collected in 2008-2009 through the Study for Monitoring Antimicrobial Resistance Trends (SMART), an ongoing global longitudinal surveillance study that has been monitoring the susceptibility of Gram-negative aerobic pathogens from intra-abdominal infections since 2002. To achieve our goals, we used a nucleic acid based-microarray technology (Check-KPC ESBL) and PCR-sequencing. To date, this microarray has performed with a sensitivity and specificity between 95 and 100% (8, 10, 21, 28).
MATERIALS AND METHODS
Bacterial strains.
Enterobacteriaceae isolates (2008–2009) were collected through the SMART program. Phenotypic ESBL testing was done by broth microdilution according to CLSI guidelines, with a ≥3-doubling-dilution decrease in the MIC of ceftazidime or cefotaxime in the presence of clavulanic acid compared to the antibiotics alone (7). The disk diffusion confirmation test, using both cefotaxime and ceftazidime alone (30 μg) and in combination with clavulanate disks (30/10 μg), was performed for verification of the ESBL phenotype (7). In this collection, 1,093 isolates with ESBL-producing phenotypes were tested. In addition, 59 non-ESBL-producing isolates, including Escherichia coli ATCC 25922, were used as negative controls. Eight Escherichia coli or Klebsiella pneumoniae strains, representing the ESBLs targeted by the Check-Points method (blaSHV-12, blaTEM-52, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaCTX-M-8, blaCTX-M-25, and blaKPC-2), were used as positive controls.
The 1,093 ESBL-positive isolates included Escherichia coli (n = 730; 66.8%), Klebsiella pneumoniae (n = 340; 31.1%), Klebsiella oxytoca (n = 12; 1.1%), and Proteus mirabilis (n = 11; 1%). Isolates were collected from intra-abdominal infections in 35 countries from seven regions: Africa (n = 7; 0.6%), Asia (n = 416, 38%), Europe (n = 300; 27.5%), Latin America (n = 229; 21%), the Middle East (n = 23; 2.1%), North America (n = 66; 6%), and the South Pacific (n = 52; 4.8%) (Fig. 1).
Fig 1.
Worldwide distribution of the 1,093 clinical isolates tested.
DNA isolation.
Whole-genomic DNA was extracted from overnight colonies grown on blood agar (Remel, Lenexa, KS) using a QIAamp DNA Mini kit and a QIAcube instrument (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Nucleic acid-based microarray.
The Check-KPC ESBL microarray was performed according to the manufacturer's instructions (Check-Points) as previously described (8, 10, 21).
Amplification and DNA sequencing of the ESBL and KPC genes.
PCR was performed on the blaESBL and blaKPC genes identified by microarray on an ABI 9700 thermocycler (Applied Biosystems, Carlsbad, CA). bla genes of the TEM, SHV, CTX-M, and KPC types were amplified as previously described (18, 24, 32, 36, 39). PCR was carried out with a Fast Cycling PCR kit (Qiagen, Valencia, CA). Purification of the PCR products was performed using Exo-SAP-IT (USB, Cleveland, OH). PCR-amplified fragments were sequenced using an ABI 3730XL DNA analyzer (Applied Biosystems). Nucleotide sequences were analyzed with SeqScape v. 7.0 (Applied Biosystems) and compared to sequences available at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).
Statistical analysis.
Fisher's exact test (two-tailed) was used to evaluate differences in blaCTX-M, blaSHV, and blaTEM carriage rates between E. coli and K. pneumoniae isolates. A P value of ≤0.05 was considered statistically significant.
Nucleotide sequence accession numbers.
The complete sequences of the novel blaSHV and blaKPC β-lactamase genes have been deposited in the GenBank database under the accession numbers GU827715 (blaSHV-129) and HM066995 (blaKPC-11).
RESULTS
Detection and identification of blaESBL genes of the TEM, SHV, and CTX-M types.
Of the 1,093 isolates from diverse regions that possessed an ESBL phenotype, 1,041 were positive using the microarray for blaESBL and/or blaKPC genes. All the blaSHV-, blaTEM-, and blaCTX-M-type ESBLs detected by the array were positive by PCR. For all the remaining isolates (n = 52; 4.8%), which were negative by this method, the microarray images were analyzed visually and were suspected to be positive because of the presence of a weak signal. Indeed, they were positive for blaCTX-M-1 (n = 50), blaTEM-11 (n = 1), and blaSHV-12 (n = 1) by PCR-sequencing. Of the 1,041 isolates with a positive microarray result, 9 were blaESBL negative but blaKPC positive.
We were able to compare only the positive results obtained by the microarray (n = 1,041) with the sequencing results, since the PCR amplifications were not performed on those with negative results, unless a weak signal was observed. Positive microarray results were highly comparable (94.2% concordance) to those of PCR-sequencing of the entire genes for isolates harboring one or more than one ESBL gene. The sequencing failed to detect a blaESBL gene and detected blaSHV-1, blaSHV-11, or blaTEM-1 in phenotypically blaSHV and blaTEM ESBL-positive isolates, respectively (n = 11) (Table 1).
Table 1.
Correlation between the microarray results and the sequencing results for the 1,093 clinical isolates tested
| β-Lactamase genotype based on microarray results (n = 1,093a) | No. (%) of isolates with concordant results (n = 1,030, 94.2%) | Sequencing results |
|
|---|---|---|---|
| β-Lactamase genes | Non-ESBL β-lactamase genes (n = 11) | ||
| SHV (n = 133a) | 126 (94.7) | SHV-2, -2A, -5, -7, -12, -45, -55, -102, -129 | SHV-1, -11b (n = 6) |
| TEM (n = 8a) | 7 (87.5) | TEM-11, -12, -26, -52, -92 | |
| CTX-M-1 group (n = 698a) | 648 (92.8) | CTX-M-1, -3, -12, -15, -22, -28, -30, -55, -61, -79 | |
| CTX-M-2 group (n = 31) | 31 (100) | CTX-M-2 | |
| CTX-M-9 group (n = 147) | 147 (100) | CTX-M-9, -14, -24, -27, -65 | |
| CTX-M-8/25 group (n = 3) | 3 (100) | CTX-M-8, -39 | |
| SHV, -CTX-M-1 group (n = 21) | 20 (95.2) | SHV-12 + CTX-M-3, -15, -28, -79 | |
| SHV-7 + CTX-M-15 | |||
| SHV-2 + CTX-M-79 | |||
| SHV-2A + CTX-M-15 | |||
| SHV-5 + CTX-M-15 | |||
| SHV-31 + CTX-M-15 | |||
| SHV-55 + CTX-M-15 | |||
| SHV-120 + CTX-M-15 | |||
| CTX-M-15 | SHV−28b (n = 1) | ||
| SHV, -CTX-M-2 group (n = 2) | 2 (100) | SHV-12 + CTX-M-2 | |
| SHV, -CTX-M-9 group (n = 5) | 5 (100) | SHV-12 + CTX-M-9 | |
| SHV-12 + CTX-M-14 | |||
| SHV-5 + CTX-M-9 | |||
| SHV, -CTX-M-8/25 group (n = 1) | 0 (0) | CTX-M-8 | SHV−11b (n = 1) |
| TEM, -CTX-M-1 group (n = 5) | 4 (80) | TEM-52 + CTX-M-15 | |
| TEM-52 + CTX-M-28 | |||
| CTX-M-61 | TEM−1b (n = 1) | ||
| TEM, -CTX-M-9 group (n = 1) | 0 (0) | CTX-M-14 | TEM−1b (n = 1) |
| CTX-M-1 + 9 groups (n = 7) | 7 (100) | CTX-M-15 + CTX-M-14 | |
| CTX-M-79 + CTX-M-14 | |||
| CTX-M-2 + 8/25 groups (n = 1) | 1 (100) | CTX-M-2 + CTX-M-8 | |
| SHV, -TEM, -CTX-M-1 group (n = 2) | 1 (50) | SHV-12 + TEM-52 + CTX-M-15 | |
| CTX-M-15 | SHV−1b + TEM−1b (n = 1) | ||
| SHV KPC (n = 18) | 18 (100) | SHV-12 + KPC-2, -3, -11 | |
| SHV-5 + KPC-2 | |||
| SHV, -CTX-M-1 group, -KPC (n = 1) | 1 (100) | SHV-12 + CTX-M-32 + KPC-2 | |
| KPC (n = 9) | 9 (100) | KPC-2, -3 | |
Including the weak signals not detected by the microarray.
The sequences showed none of the mutations conferring the ESBL phenotype, only the non-ESBL genes blaSHV-1 and blaSHV-11.
There were also some minor discrepancies between the mutations detected by the microarray and by sequencing for blaSHV and blaTEM genes (Table 2). Among the blaSHV- and blaTEM-possessing isolates, our results were discordant for 27 blaSHV isolates (15%) and one blaTEM isolate (6%). The mutations detected by the microarray were either not interpretable (n = 9) or misinterpreted by the software (n = 19) (Table 2).
Table 2.
Minor discrepancies between the mutations detected by the microarray and by sequencing in 28 blaESBL-positive isolatesa
| Species (no. of isolates) | No. of isolates (n = 28) | Mutations detected by microarray | β-Lactamase gene sequencing results | Comments |
|---|---|---|---|---|
| E. coli (7) and K. pneumoniae (2) | 9 | Noninterpretable | SHV12 (L35Q, G238S, E240K) | Smear signals on microarray |
| K. pneumoniae (17) | 11 | 238G, 240E, E240K | SHV12 (L35Q, G238S, E240K) | Weak signal for G238S on microarray |
| 5 | 238G, 240E, E240K | SHV5 (G238S, E240K) | ||
| 1 | 238G, 240E, E240K | SHV55 (Y7F, G238S, E240K) | ||
| K. pneumoniae (1) | 1 | 238G, G238A, 240E | SHV28 (Y7F) | Background signal for G238A on microarray |
| E. coli (1) | 1 | 104E, E104K, 164R, 238G | TEM52 (E104K, M182T, G238S) | Weak signal for G238S on microarray |
Boldface indicates mutations involved in the ESBL phenotype.
A particular shortcoming of the microarray system was the failure to detect the blaCTX-M-1 genes in 50 isolates, representing 4.6% of the 1,093 blaESBL-positive isolates. This had already been observed in previous studies (21, 28). We minimized this specific shortcoming by carefully checking the microarray visually and confirming by PCR-sequencing any weak signals not interpreted as positive by the software. However, when detected by the microarray, the blaCTX-M genes were always in accordance with the group detected, as verified by sequencing. Furthermore, the CTX-M-1 probe has recently been improved and the software updated, which should minimize or eliminate this problem in the future (4).
Detection and identification of blaKPC.
All of the 28 blaKPC-possessing isolates detected by microarray were positive for blaKPC genes by PCR, including 9 ESBL-negative isolates which had originally been phenotypically characterized as ESBL positive. The 28 isolates were typed as blaKPC-2 (n = 16; 57%), blaKPC-3 (n = 8; 29%), and a new variant, blaKPC-11 (n = 4; 14%) (GenBank accession number HM066995). blaKPC-3 differs from blaKPC-2 by a single amino acid substitution (H272Y), and blaKPC-11 also differs from blaKPC-2 by a single amino acid substitution, but at position 103 (P103L). The blaKPC-11 isolates were collected from peritoneal fluids or gastrointestinal specimens from four different patients hospitalized in the same department (Surgery General) in Greece in 2009.
Worldwide distribution of ESBL enzymes.
The 1,093 blaESBL and/or blaKPC-positive isolates were collected from 5 regions (Table 3) and 35 countries (Fig. 1). The CTX-M-1 group was the most frequent in all regions (65.4% to 100%) in E. coli isolates. In K. pneumoniae isolates, the CTX-M-1 group was also the most common (40% to 75%), except in North America, where the SHV type predominates (59%). Of the E. coli isolates possessing multiple ESBL enzymes, most were isolated in Latin America (40%) and Asia (35%). The distribution observed for K. pneumoniae isolates possessing multiple ESBL enzymes was quite similar: predominantly Asia (35%) and Latin America (31%). The CTX-M-1 group was also the most common group present in K. oxytoca and P. mirabilis (Table 3).
Table 3.
Worldwide distribution of blaESBL/KPC genes by species for the 1,093 clinical isolates tested
| Species (no. of isolates) and ESBL type | No. (%) of isolates from region |
|||||||
|---|---|---|---|---|---|---|---|---|
| Africa | Asia | Europe | Latin America | Middle East | North America | South Pacific | Total | |
| E. coli (730) | ||||||||
| SHV | 4 (1.3) | 23 (11.1) | 12 (8.1) | 1 (4.8) | 2 (7.7) | 42 | ||
| TEM | 2 (0.6) | 2 (0.9) | 1 (0.7) | 5 | ||||
| CTX-M-1 group | 3 (100) | 225 (72.6) | 142 (68.3) | 110 (73.8) | 10 (76.9) | 14 (66.6) | 17 (65.4) | 521 |
| CTX-M-2 group | 2 (0.9) | 9 (6) | 11 | |||||
| CTX-M-9 group | 72 (23.2) | 36 (17.3) | 7 (4.7) | 2 (15.4) | 6 (28.6) | 6 (23.1) | 129 | |
| CTX-M-8/25 group | 2 (1.3) | 2 | ||||||
| CTX-M-1 + 9 groups | 3 (1) | 1 (0.5) | 2 (1.3) | 6 | ||||
| CTX-M-2 + 8/25 groups | 1 (0.7) | 1 | ||||||
| SHV + CTX-M-1 group | 3 (1) | 1 (0.7) | 1 (7.7) | 5 | ||||
| SHV + CTX-M-2 group | 2 (1.3) | 2 | ||||||
| SHV + CTX-M-9 group | 1 (0.3) | 1 (0.7) | 1 (3.8) | 3 | ||||
| TEM + CTX-M-1 group | 1 (0.5) | 1 (0.7) | 2 | |||||
| TEM + CTX-M-9 group | 1 (0.5) | 1 | ||||||
| Species total | 3 | 310 | 208 | 149 | 13 | 21 | 26 | 730 |
| K. pneumoniae (340) | ||||||||
| SHV | 1 (25) | 15 (15.3) | 31 (36) | 9 (12.5) | 26 (59) | 6 (23.1) | 88 | |
| TEM | 1 (1) | 1 (1.4) | 2 | |||||
| CTX-M-1 group | 3 (75) | 62 (63.3) | 35 (40.7) | 32 (44.4) | 4 (40) | 8 (18.2) | 17 (65.4) | 161 |
| CTX-M-2 group | 20 (27.7) | 1 (2.3) | 21 | |||||
| CTX-M-9 group | 11 (11.2) | 3 (3.5) | 14 | |||||
| CTX-M-8/25 group | 1 (10) | 1 | ||||||
| CTX-M-1 + 9 groups | 1 (1.2) | 1 | ||||||
| SHV + CTX-M-1 group | 6 (6.1) | 2 (2.3) | 3 (4.2) | 1 (10) | 1 (2.3) | 2 (7.7) | 15 | |
| SHV + CTX-M-9 group | 3 (3.1) | 3 | ||||||
| SHV + CTX-M-8/25 group | 1 (1.4) | 1 | ||||||
| TEM + CTX-M-1 group | 3 (4.2) | 3 | ||||||
| SHV + TEM + CTX-M-1 group | 1 (1.4) | 1 (3.8) | 2 | |||||
| SHV + KPC | 13 (15.1) | 1 (10) | 4 (9.1) | 18 | ||||
| SHV + CTX-M-1 + KPC | 1 (1.2) | 1 | ||||||
| KPC | 2 (2.8) | 3 (30) | 4 (9.1) | 9 | ||||
| Species total | 4 | 98 | 86 | 72 | 10 | 44 | 26 | 340 |
| Klebsiella oxytoca (12) | ||||||||
| SHV | 2 (50) | 1 (100) | 3 | |||||
| CTX-M-1 group | 3 (60) | 2 (50) | 2 (100) | 7 | ||||
| CTX-M-9 group | 2 (40) | 2 | ||||||
| Species total | 5 | 4 | 2 | 1 | 12 | |||
| Proteus mirabilis (11) | ||||||||
| TEM | 1 (50) | 1 | ||||||
| CTX-M-1 group | 2 (66.7) | 1 (50) | 6 (100) | 9 | ||||
| CTX-M-9 group | 1 (33.3) | 1 | ||||||
| Species total | 3 | 2 | 6 | 11 | ||||
| Total | 7 | 416 | 300 | 229 | 23 | 66 | 52 | 1,093 |
The distribution by species (Table 3) shows that 93.6% (n = 683) of E. coli carry blaCTX-M genes, either alone (91.8%) or in combination with other genes, such as blaSHV or blaTEM (1.8%). Furthermore, 65.6% (n = 223) of K. pneumoniae isolates also carry blaCTX-M genes, either alone (58.2%) or in combination with other genes, such as blaSHV and/or blaTEM (7.1%) and blaKPC genes (5.6%). However, 37.6% (n = 128) of K. pneumoniae ESBL producers carry a SHV-type blaESBL gene, while only 7.1% of E. coli (n = 52) ESBL producers carry this type. Overall, the distribution of blaCTX-M and blaSHV genes was significantly different between E. coli and K. pneumoniae (Fisher's exact test, P < 0.0001). Finally, there was no significant difference for blaTEM genes: 1.1% of E. coli and 2% of K. pneumoniae isolates were blaTEM positive (Fisher's exact test, P = 0.2634). Overall, among the blaCTX-M-possessing isolates, 67% possessed blaCTX-M-15, with these isolates being predominantly E. coli (73.8%) and K. pneumoniae isolates (24.7%). Also, 14% possessed blaCTX-M-14, predominantly E. coli isolates (86.5%). blaSHV-12 was identified in 60% of blaSHV ESBL-carrying isolates, and 19% carried blaSHV-5, predominantly K. pneumoniae isolates (see Fig. S1 in the supplemental material). In Table 4, the diversity and complexity of ESBL enzyme variants are summarized.
Table 4.
Distribution of blaESBL/KPC genes among the 1,093 clinical isolates collected worldwide
| Species (no. of isolates) | Type of ESBL or KPC (no. [%] of isolates) |
||||
|---|---|---|---|---|---|
| TEM | SHV | CTX-M | Mixed ESBLs | KPCs (±ESBLs) | |
| E. coli (730) | TEM-52 (3 [0.4]) | SHV-12 (32 [4.4]) | CTX-M-15 (451 [61.8]) | SHV-12 + CTX-M-15 (4 [0.5]) | |
| TEM-12 (1 [0.1]) | SHV-5 (5 [0.7]) | CTX-M-14 (101 [13.8]) | SHV-12 + CTX-M-2 (2 [0.3]) | ||
| TEM-11 (1 [0.1]) | SHV-2 (2 [0.3])) | CTX-M-61 (23 [3.1]) | SHV-12 + CTX-M-79 (1 [0.1]) | ||
| SHV-2A (1 [0.1]) | CTX-M-2 (11 [1.5]) | SHV-12 + CTX-M-9 (1 [0.1]) | |||
| SHV-102 (1 [0.1]) | CTX-M-79 (10 [1.4]) | SHV-5 + CTX-M-14 (1 [0.1]) | |||
| SHV-129b (1 [0.1]) | CTX-M-27 (10 [1.4]) | SHV-7 + CTX-M-15 (1 [0.1]) | |||
| CTX-M-3 (8 [1.1]) | TEM-52 + CTX-M-15 (1 [0.1]) | ||||
| CTX-M-9 (8 [1.1]) | TEMa + CTX-M-14 (1 [0.1]) | ||||
| CTX-M-24 (8 [1.1]) | TEMa + CTX-M-61 (1 [0.1]) | ||||
| CTX-M-28 (8 [1.1]) | CTX-M79 + CTX-M14 (3 [0.4]) | ||||
| CTX-M-55 (7 [0.9]) | CTX-M15 + CTX-M14 (3 [0.4]) | ||||
| CTX-M-1 (6 [0.8]) | CTX-M-2 + CTX-M-8 (1 [0.1]) | ||||
| CTX-M-22 (6 [0.8]) | |||||
| CTX-M-8 (2 [0.3]) | |||||
| CTX-M-65(2 [0.3]) | |||||
| CTX-M-12 (1 [0.1]) | |||||
| CTX-M-30 (1 [0.1]) | |||||
| K. pneumoniae (340) | TEM-12 (1 [0.3]) | SHV-12 (41 [12]) | CTX-M-15 (137 [40.3]) | SHV-12 + CTXM-15 (3 [0.9]) | KPC-2 (3 [0.9]) |
| TEM-26 (1 [0.3]) | SHV-5 (22 [6.5]) | CTX-M-2 (20 [5.9]) | SHV-12 + CTX-M-9 (2 [0.6]) | KPC-3 (6 [1.8]) | |
| SHV-2 (8 [2.3]) | CTX-M-14 (13 [3.8]) | SHV-12 + CTX-M-28 (1 [0.3]) | SHV-12 + KPC-2 (12 [3.5]) | ||
| SHV-2A (5 [1.5]) | CTX-M-28 (7 [2]) | SHV-12 + CTX-M-3 (1 [0.3]) | SHV-12 + KPC-3 (2 [0.6]) | ||
| SHV-55 (4 [1.2]) | CTX-M-3 (6 [1.7]) | SHV-12 + TEM-52 + CTX-M-15 (1 [0.3]) | SHV-12 + KPC-11 (4 [1.2]) | ||
| SHV-45 (1 [0.3]) | CTX-M-22 (3 [0.9]) | SHV-5 + CTX-M-15 (4 [1.2]) | SHV-12 + CTX-M-32 + KPC-2 (1 [0.3]) | ||
| SHV-7 (1 [0.3]) | CTX-M-55 (3 [0.9]) | SHV-5 + CTX-M-9 (1 [0.3]) | |||
| SHVa (6 [1.8]) | CTX-M-61 (2 [0.6]) | SHV-2 + CTX-M-79 (1 [0.3]) | |||
| CTX-M-9 (1 [0.3]) | SHV-2A + CTX-M-15 (1 [0.3]) | ||||
| CTX-M-12 (1 [0.3]) | SHV-28 + CTX-M-15 (1 [0.3]) | ||||
| CTX-M-27 (1 [0.3]) | SHV-31 + CTX-M-15 (1 [0.3]) | ||||
| CTX-M-30 (1 [0.3]) | SHV-55 + CTX-M-15 (1 [0.3]) | ||||
| CTX-M-39 (1 [0.3]) | SHV-120 + CTX-M-15 (1 [0.3]) | ||||
| CTX-M-79 (1 [0.3]) | TEM-52 + CTX-M-15 (2 [0.6]) | ||||
| TEM-52 + CTX-M-28 (1 [0.3]) | |||||
| CTX-M-15 + CTX-M-14 (1 [0.3]) | |||||
| SHVa+TEMa+CTX-M-15 (1 [0.3]) | |||||
| SHVa+CTX-M-8 (1 [0.3]) | |||||
| K. oxytoca (12) | SHV-5 (1 [8.3]) | CTX-M-14 (2 [16.6]) | |||
| SHV-7 (1 [8.3]) | CTX-M-15 (4 [33.3]) | ||||
| SHV-12 (1 [8.3]) | CTX-M-22 (1 [8.3]) | ||||
| CTX-M-30 (2 [16.6]) | |||||
| P. mirabilis (11) | TEM-92 (1 [9.1]) | CTX-M-3 (1 [9.1]) | |||
| CTX-M-14 (1 [9.1]) | |||||
| CTX-M-15 (5 [45.5]) | |||||
| CTX-M-22 (1 [9.1]) | |||||
| CTX-M-28 (1 [9.1]) | |||||
| CTX-M-61 (1 [9.1]) | |||||
Unsuccessful sequencing of the ESBL gene.
New variant reported in this study.
It is important to note that our scheme, microarray plus PCR-sequencing, was able to fully characterize 72 isolates in 3.5 days, compared to the 6 days required by the conventional method, and reduced the number of PCR-sequencing reactions required. As an example, for a K. pneumoniae isolate positive for blaCTX-M-15 by microarray, the CTX-M-1 group PCR was the only PCR performed. If the conventional method had been used, PCRs for the SHV, TEM, and CTX-M (CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-8, and CTX-M-25) groups would have been performed. This means that a single PCR-sequencing reaction instead of seven reactions was required.
While the 1,093 phenotypically ESBL-positive isolates were being characterized, a novel blaSHV gene was detected in an E. coli isolate: blaSHV-129. This isolate was collected from an abscess specimen from a patient hospitalized in Italy in 2008. The substitutions detected by the microarray in this gene were G238S and E240K. Indeed, the blaSHV-129 gene encodes five amino acid substitutions (L35Q, G238S, E240K, R275L, and N276D). Three of these substitutions (L35Q, R275L, and N276D) were determined only by sequencing, since they are not included in the microarray system, while G238S and E240K were confirmed by sequencing.
DISCUSSION
Detection of ESBL-mediated and carbapenem resistance in Gram-negative bacilli is necessary for directed therapy, improving clinical outcomes, and limiting the spread of these multidrug-resistant organisms (6). The heterogeneous nature of resistance among ESBL- and KPC-producing isolates makes detection more complicated if it is based on susceptibility testing alone (23, 33). These difficulties may delay appropriate patient treatment, as well as response to an outbreak. In our study, we addressed the current status of ESBLs and KPCs in Gram-negative bacteria.
Current status of ESBLs.
As stated above, few studies have been performed examining the molecular epidemiology of large numbers of clinical isolates possessing an ESBL phenotype. A previous analysis examining K. pneumoniae isolates was among the first to give a snapshot of the SHV and TEM sequence variability of ESBLs found in bloodstream isolates from patients on different continents at a single point in time (1996 to 1997) (27). This was a necessary first step in the quest to understand how the genotypes of these complex antibiotic resistance phenotypes emerge in the clinical setting. A major finding at that time was that SHV-type ESBLs were by far the most dominant ESBL type and that blaCTX-M was an emerging resistance determinant of significant importance. At the time of that study, it was speculated that the types of β-lactamases produced by K. pneumoniae may also be evolving.
Our large-scale survey reports that the molecular epidemiology of ESBLs in E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis is evolving, as was the status of K. pneumoniae ESBL-producing organisms from 1996–1997. We found that E. coli is the most common pathogen harboring ESBLs and that blaCTX-M-15, blaCTX-M-14, and blaSHV-12 are the most common ESBLs detected. We also found strains with novel ESBL genotypes. Among the 1,093 isolates identified that were ESBLs by phenotype, we identified one novel blaSHV gene (SHV-129) and one novel blaKPC gene (KPC-11). Although there are no molecular or epidemiological benchmarks against which to readily compare this rate of novel gene discovery, we believe this represents a high frequency of emergence; a rate of one novel ESBL/KPC bla gene in 500 ESBL-positive strains is troubling. As noted previously, analysis of the plasmid background and genetic context of these strains may provide the keys to understanding why these phenotypes are becoming so prevalent.
Impact of rapid testing with microarrays.
During the last 20 years, alternative molecular strategies for ESBL determination have been proposed (1, 19, 16). More recently, new technologies, such as real-time PCR, pyrosequencing, and denaturing high-performance liquid chromatography (dHPLC) (3, 13, 38), have been employed, but they still remain unsuitable for the clinical laboratory. Conventional PCR remains the most convenient and cost-effective method to confirm phenotypic results for routine laboratories. However, large-scale epidemiological studies can become time-consuming and expensive when the classical PCR-sequencing method is used. Fast and accurate methods are crucial for monitoring the constantly changing epidemiology of β-lactamases.
The nucleic acid-based microarray combined with PCR-sequencing was able to confirm the presence of a blaESBL gene, differentiate blaESBL from blanon-ESBL genes, and precisely identify the ESBL genotype. Also, by indicating the presence of blanon-ESBL and blaESBL and the mutations involved, microarrays facilitate the sequencing analysis of the double peaks in blaESBL-carrying K. pneumoniae or E. coli isolates harboring a non-ESBL gene (blaSHV-1 or blaTEM-1, respectively) along with ESBL genes.
The discrepancies observed between the two methods were due either to the microarray probe or software or to sequencing issues. These data support the previous observation that DNA sequencing of PCR amplification products in certain isolates fails to accurately detect all ESBL genes (10, 21). However, a worrisome issue already observed by Platteel et al. was the detection of the blaCTX-M-1-positive isolates, since this is the most prevalent group worldwide (28). Fortunately, this issue seems to have been resolved in all Check-Points kits; as an example, Bogaerts et al. reported 100% specificity and sensitivity of the Check-Points ESBL/AmpC/KPC/NDM-1 (Check-MDR CT101) kit for the blaCTX-M genes (4).
Conclusions.
Microarray combined with PCR-sequencing appears to be an efficient strategy for examining quickly the molecular epidemiology of resistance genes, especially where large collections of isolates require characterization. Nevertheless, we must caution that for TEM and SHV β-lactamases, new variants may not be detected by microarrays, since oligonucleotide probes do not cover all TEM- and SHV-type β-lactamases (8). However, the flexibility of this system is an important advantage, since new primers and probes can be included depending on current molecular epidemiology (4, 20). The promising results obtained in previous evaluation studies (8, 10, 21, 28, 35) were confirmed in our large survey and emphasize the performance of the microarray as a reliable tool to guide PCR-sequencing.
We close with the following observation: worldwide epidemiological surveillance studies examining ESBL and carbapenemase genes are essential not only to monitor the dissemination of these enzymes but also to monitor the emergence of new variants that may spread globally (e.g., SHV-129, KPC-11, CTX-M-15, or NDM-1). The analysis summarized herein gives an important assessment of the current state of ESBLs throughout the world. Nucleic acid microarrays performed on a large number of clinical isolates demonstrate an application used in combination with PCR-sequencing for complete characterization of β-lactamase genes in epidemiological studies. Furthermore, the flexibility of the system has been extended to other important genes, such as blaAmpC and blaMBLs, including blaNDM-1 (4, 20, 37). We note that the molecular background of ESBL-producing pathogens found within the past 5 years has grown significantly in complexity (27). Studies are required to measure how the emerging ESBL problem is impacting the delivery of care.
Supplementary Material
ACKNOWLEDGMENTS
The Study for Monitoring Antimicrobial Resistance Trends is funded by Merck Research Laboratories, Inc. R.A.B. is funded by The Veterans Affairs Merit Review Program, the National Institutes of Health (RO1AI063517-07, RO1AI072219-05), and VISN 10 Geriatric Research Education and Clinical Center (GRECC).
We thank Johann Pitout (University of Calgary, Calgary, Canada) for providing control strains, and Check-Points for the technical and helpful support. We gratefully acknowledge contributions from the current participants in the SMART program.
We have no conflict of interest to declare.
Footnotes
Published ahead of print 8 February 2012
Supplemental material for this article may be found at http://jcm.asm.org/.
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