Abstract
Of 1,456 strains isolated from 2001 to 2003 demonstrating resistance to either oxyimino-cephalosporin, 317 strains, isolated in 57 of 132 clinical facilities, were found to harbor blaCTX-M genes by PCR. Fifty-seven, 161, and 99 strains harbored blaCTX-M genes belonging to the blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 clusters, respectively.
In recent years, CTX-M-type β-lactamases have been recognized as a growing family possessing a high level of hydrolyzing activities, especially against cefotaxime (CTX) and ceftriaxone. Nearly 40 variants of the CTX-M-type enzymes have been identified (2, 4, 13, 25) and registered to date (http://www.lahey.org/studies/other.asp#table_1). Further proliferation of CTX-M-type β-lactamase-producing gram-negative bacteria has become a great concern (6), since a large number of nosocomial outbreaks caused by such bacteria have so far been recognized and reported in various medical facilities in many countries (1, 3, 5, 7-9, 19, 21).
In Japan, FEC-1 and Toho-1 were initially identified (12, 15) and were later included in CTX-M-type enzymes. Since then, various strains that produce a Toho-1-like β-lactamase have been identified in Japanese clinical settings (26, 28). Almost all of them, however, were found to be CTX-M-2 by sequence analyses (N. Shibata, et al. Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-2235, 2001). However, the trends for several CTX-M-type β-lactamases other than CTX-M-2 have remained unclear. In the present study, we investigated the molecular types of CTX-M-type β-lactamases produced by nosocomial gram-negative bacilli isolated in Japanese clinical facilities using PCR methods.
From January 2001 to December 2003, 1,456 gram-negative bacterial isolates demonstrating resistance to oxyimino-cephalosporins were submitted from 132 hospitals to the reference laboratory at our institute. These strains were then subjected to screening for β-lactamases, including TEM- and SHV-derived extended-spectrum β-lactamases (ESBLs), CTX-M-type β-lactamases, AmpC- and CMY-type class C cephalosporinases and cephamycinases, and class B metallo-β-lactamases (MBLs). The strains were checked for ESBL production by the double-disk diffusion synergy test recommended by the CLSI (formerly the NCCLS) (18). The MICs of ceftazidime (CAZ) and CTX for the clinical isolates were determined by the agar dilution method recommended by the CLSI guidelines. When a clinical isolate demonstrated resistance to either oxyimino-cephalosporin, the strain was then subjected to PCR analyses for detection of blaCTX-M genes. PCR analysis was performed by the method reported previously (27). The four sets of PCR primers used for detection of blaCTX-M genes in the present study were as follows: primers CTX-M-1-F (5′-GCT GTT GTT AGG AAG TGT GC-3′) and CTX-M-1-R (5′-CCA TTG CCC GAG GTG AAG-3′), primers CTX-M-2-F (5′-ACG CTA CCC CTG CTA TTT-3′) and CTX-M-2-R (5′-CCT TTC CGC CTT CTG CTC-3′), primers CTX-M-8-F (5′-CGG ATG ATG CTA ATG ACA AC-3′) and CTX-M-8-R (5′-GTC AGA TTG CGA AGC GTC-3′), and primers CTX-M-9-F (5′-GCA GAT AAT ACG CAG GTG-3′) and CTX-M-9-R (5′-CGG CGT GGT GGT GTC TCT-3′). Only one strain was selected from an individual patient and subjected to the PCR test.
As shown in Table 1, the inhibition patterns by combination of the double-disk diffusion synergy test for ESBL detection and the sodium mercaptoacetic acid (SMA) disk test for MBL detection were classified into four groups. Of 1,456 strains tested, 59 were resistant only to CAZ and susceptible to clavulanic acid. It was speculated that these strains produce mainly SHV- or TEM-derived ESBLs, because SHV-12-producing strains have been prevalent in Japan (27). On the other hand, 276 strains showed resistance to CTX but were susceptible to CAZ. The MIC of CTX was significantly decreased in the presence of clavulanic acid. It was speculated that these strains chiefly produce CTX-M-type β-lactamases. Five hundred forty-eight isolates demonstrated resistance to both CAZ and CTX; but the inhibitory effect of clavulanic acid was not clear in these strains, and the production of MBL was suggested, because the MICs of CAZ and CTX were reduced in the presence of SMA, which is a specific inhibitor of metallo-β-lactamase (23). The remaining 573 strains, which demonstrated resistance to either of the oxyimino-cephalosporins, did not become susceptible to these agents in the presence of SMA, suggesting the production of some AmpC-type enzymes, including plasmid-mediated CMY-type enzymes.
TABLE 1.
Results of screening by double-disk diffusion synergy tests
| Bacterial species | Pattern of double-disk diffusion synergy test
|
Total no. of strains tested | |||
|---|---|---|---|---|---|
| Resistant to CAZ and susceptible to clavulanic acid (no. of strains) | Resistant to CTX and susceptible to clavulanic acida | Resistant to CAZ and CTX and susceptible to SMAa | Resistant to either oxyimino-cephalosporin and not susceptible to SMA | ||
| Escherichia coli | 33 | 157/157 | 7/24 | 4/4 | 218 |
| Proteus mirabilis | 0 | 71/71 | 0/1 | 0/0 | 72 |
| Klebsiella pneumoniae | 15 | 42/42 | 7/31 | 1/2 | 90 |
| Klebsiella oxytoca | 4 | 5/5 | 1/3 | 0/2 | 14 |
| Serratia marcescens | 7 | 0/0 | 0/65 | 10/77 | 149 |
| Enterobacter cloacae | 0 | 0/0 | 2/11 | 1/20 | 31 |
| Enterobacter aerogenes | 0 | 0/0 | 0/2 | 1/8 | 10 |
| Citrobacter freundii | 0 | 0/0 | 0/4 | 2/15 | 19 |
| Citrobacter koseri | 0 | 0/0 | 0/0 | 1/1 | 1 |
| Providencia rettgeri | 0 | 1/1 | 0/2 | 0/0 | 3 |
| Acinetobacter baumannii | 0 | 0/0 | 1/49 | 3/40 | 89 |
| Other bacterial speciesd | 0 | 0/0 | 0/356 | 0/404 | 760 |
| Totale | 59 | 276/276 | 18/548b | 23/573c | 1,456 |
The data represent the number of blaCTX-M-positive strains by PCR/total number of strains demonstrating each inhibition pattern and subjected to PCR.
Strains that produce metallo-β-lactamase are included.
Strains that produce plasmid-mediated CMY-type cephalosporinase or chromosomal AmpC hyperproducers are included.
Pseudomonas spp., Alcaligenes spp., Achromobacter spp., and Burkholderia spp. demonstrating resistance to ceftazidime or cefotaxime were included; but Stenotrophomonas spp. and Chryseobacterium spp. that produce intrinsic metallo-β-lactamase were excluded.
Out of the total number of strains being subjected to PCR analysis (1,397; represented in columns 2, 3, and 4), 317 were found to be blaCTX-M positive.
Of 1,397 strains subjected to the PCR analyses, 317 strains were suggested to harbor blaCTX-M genes. Of these strains, 57 appeared to carry genes of the blaCTX-M-1 group, including blaCTX-M-1, blaCTX-M-3, and blaCTX-M-15, as shown in Table 2. Moreover, 161 strains were suggested to harbor the genes encoding the CTX-M-2 group of enzymes, such as CTX-M-2, CTX-M-20, and CTX-M-31. Furthermore, 99 strains appeared to carry the genes for the CTX-M-9 group of enzymes, such as CTX-M-9, CTX-M-14, and CTX-M-16. No strain harboring genes for the CTX-M-8 or the CTX-M-25 group of enzymes was found among the strains tested.
TABLE 2.
Number of strains that produce CTX-M-type β-lactamases as detected by PCR
| Bacterial species | No. of strains by the following PCR type:
|
Total | ||
|---|---|---|---|---|
| CTX-M-1 groupa | CTX-M-2 groupb | CTX-M-9 groupc | ||
| Escherichia coli | 33 | 46 | 89 | 168 |
| Proteus mirabilis | 0 | 71 | 0 | 71 |
| Klebsiella pneumoniae | 10 | 31 | 9 | 50 |
| Klebsiella oxytoca | 2 | 3 | 1 | 6 |
| Serratia marcescens | 9 | 1 | 0 | 10 |
| Enterobacter cloacae | 0 | 3 | 0 | 3 |
| Enterobacter aerogenes | 1 | 0 | 0 | 1 |
| Citrobacter freundii | 2 | 0 | 0 | 2 |
| Citrobacter koseri | 0 | 1 | 0 | 1 |
| Providencia rettgeri | 0 | 1 | 0 | 1 |
| Acinetobacter baumannii | 0 | 4 | 0 | 4 |
| Total | 57 | 161 | 99 | 317 |
The PCR primers used can detect genes for CTX-M-1 and several variants, such as CTX-M-3 and CTX-M-15.
The PCR primers used can detect genes for CTX-M-2 and several variants, such as CTX-M-20 and CTX-M-31.
The PCR primers used can detect genes for CTX-M-9 and several variants, such as CTX-M-14 and CTX-M-16.
As shown in Table 3, strains that harbored genes for the CTX-M-type enzymes were isolated from 57 of 132 hospitals across Japan, except for the Hokkaido region, throughout the 3-year investigation period. Fourteen and 24 strains that harbored genes for the CTX-M-1 group of enzymes were identified in 7 and 10 hospitals located in the Kanto and Chubu regions, respectively (Table 3). However, no strain harboring genes for the CTX-M-1 group of enzymes were found in the Chugoku and Shikoku regions (Table 3). In 22 of 57 hospitals, genes for multiple CTX-M-type β-lactamases belonging to different groups were identified during the investigation period (Fig. 1). Interestingly, genes for all three groups of CTX-M-type enzymes were identified in 7 of 57 hospitals (Fig. 1; Table 3).
TABLE 3.
Bacterial species that produce each group of CTX-type β-lactamases
| Region | PCR type | Bacterial species (no. of isolates) | Hospital (no. of isolates) |
|---|---|---|---|
| Hokkaido (0a/7b) | None | None | |
| Tohoku (4/17) | CTX-M-1 | K. pneumoniae (2c) | B4 (2c) |
| CTX-M-2 | E. coli (1) | B1 (1) | |
| P. mirabilis (10) | B4 (10) | ||
| CTX-M-9 | E. coli (6) | B2 (1), B3 (4), B4 (1) | |
| Kanto (9/26) | CTX-M-1 | E. coli (7) | C1 (1), C3 (2), C9 (4) |
| K. pneumoniae (6) | C2 (2), C6 (1), C7 (3) | ||
| K. oxytoca (1) | C8 (1) | ||
| CTX-M-2 | P. mirabilis (28) | C4 (9), C5 (19) | |
| A. baumannii (3) | C5 (3) | ||
| CTX-M-9 | K. pneumoniae (1) | C7 (1) | |
| E. coli (11) | C2 (1), C3 (1), C4 (1), C7 (4), C8 (4) | ||
| Chubu (22/37) | CTX-M-1 | E. coli (12) | D2 (1), D3 (5), D6 (3), D7 (1), D20 (1), D22 (1) |
| K. pneumoniae (2) | D1 (1), D20 (1) | ||
| C. freundii (2) | D18 (2) | ||
| E. aerogenes (1) | D19 (1) | ||
| S. marcescens (5) | D18 (5) | ||
| CTX-M-2 | E. coli (29) | D5 (1), D6 (2), D8 (1), D13 (4), D14 (1), D15 (5), D18 (1), D20 (14) | |
| K. pneumoniae (21) | D20 (20), D22 (1) | ||
| K. oxytoca (3) | D6 (1), D15 (1), D20 (1) | ||
| P. mirabilis (17) | D14 (4), D16 (11), D17 (1), D18 (1) | ||
| S. marcescens (1) | D20 (1) | ||
| E. cloacae (3) | D18 (1), D20 (2) | ||
| A. baumannii (1) | D20 (1) | ||
| CTX-M-9 | E. coli (34) | D4 (1), D5 (1), D6 (4), D7 (4), D8 (4), D9 (3), D10 (1), D11 (1), D12 (1), D14 (1), D16 (4), D18 (1), D20 (3), D21 (5) | |
| K. pneumoniae (4) | D12 (4) | ||
| K. oxytoca (1) | D12 (1) | ||
| Kinki (10/19) | CTX-M-1 | E. coli (6) | E5 (4), E7 (1), E10 (1) |
| K. oxytoca (1) | E4 (1) | ||
| S. marcescens (4) | E1 (4) | ||
| CTX-M-2 | E. coli (8) | E3 (1), E5 (6), E8 (1) | |
| K. pneumoniae (6) | E5 (6) | ||
| P. mirabilis (15) | E2 (1), E5 (14) | ||
| P. rettgeri (1) | E8 (1) | ||
| CTX-M-9 | E. coli (11) | E2 (2), E3 (1), E5 (6), E6 (1), E9 (1) | |
| K. pneumoniae (2) | E2 (1), E5 (1) | ||
| Chugoku (5/13) | CTX-M-2 | E. coli (3) | F2 (2), F5 (1) |
| K. pneumoniae (2) | F3 (2) | ||
| CTX-M-9 | E. coli (8) | F1 (4), F4 (1), F5 (3) | |
| Shikoku (3/5) | CTX-M-2 | E. coli (1) | G2 (1) |
| C. koseri (1) | G3 (1) | ||
| CTX-M-9 | E. coli (15) | G2 (15) | |
| K. pneumoniae (2) | G1 (1), G2 (1) | ||
| Kyushu and Okinawa (4/8) | CTX-M-1 | E. coli (8) | H1 (1), H2 (6), H3 (1) |
| CTX-M-2 | E. coli (4) | H1 (1), H4 (3) | |
| K. pneumoniae (2) | H4 (2) | ||
| P. mirabilis (1) | H2 (1) | ||
| CTX-M-9 | E. coli (4) | H1 (3), H2 (1) | |
| Total (57/132) |
Number of medical facilities where blaCTX-M-harboring strains were detected.
Number of medical facilities that submitted strains to our laboratory.
Number of clinical isolates harboring blaCTX-M gene.
FIG. 1.
Clinical facilities where multiple blaCTX-M genes belonging to different genetic clusters were identified. Facilities where multiple bacterial species that bear blaCTX-M genes were isolated are also added. The numbers in parentheses demonstrate the number of clinical isolates of each bacterial species.
After the first description of Toho-1 in Japan in 1995, several outbreaks caused by CTX-M-type β-lactamase producers have been reported in there (17, 26, 28). In the present investigation, it became clear that gram-negative nosocomial bacilli producing the CTX-M-1, CTX-M-2, or CTX-M-9 group of enzymes have already been dispersed in various clinical settings in Japan, although strains that produce TEM- or SHV-derived ESBLs are not predominant to date.
Recently, the CTX-M-1 group of enzymes, such as CTX-M-3 and CTX-M-15, have emerged in Europe and Asia (3, 8-10, 14, 22, 28). In the present study, we also identified the genes for the CTX-M-1 group of enzymes in various bacterial species, including Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, and Klebsiella oxytoca, in addition to Providencia rettgeri, Citrobacter freundii, Citrobacter koseri, and Enterobacter cloacae. This finding may be suggestive of the lateral transfer of very similar plasmids bearing blaCTX-M genes among different bacterial species. Actually, probable nosocomial transmissions of CTX-M-producing bacterial strains were suspected in several medical facilities, as shown in Fig. 1 and Table 3. Especially in hospitals D18, D20, and E5, all three groups of genes for CTX-M enzymes were identified; and genes for CTX-M-type enzymes were detected in various gram-negative bacterial species, suggesting the horizontal transfer of the blaCTX-M genes among different bacterial species. Interestingly, all 71 Proteus mirabilis strains were identified as CTX-M-2 producers, and they were isolated in widely separate medical facilities located far apart in Japan, implying a close relatedness between CTX-M-2 and P. mirabilis in Japanese clinical environments. The plasmids carrying blaCTX-M-2 may be very adaptive for P. mirabilis, which may either serve as a reservoir for plasmids carrying blaCTX-M-2 gene (16, 17) or have preferentially accepted blaCTX-M-2 genes from some environmental Kluyvera spp. (11, 20). Comparative analyses of plasmids that bear the blaCTX-M-2 gene would provide a clue to elucidate the relatedness and origins of the plasmids.
The CTX-M-9 group of enzymes, including CTX-M-14, have so far been found worldwide in the species belonging to the family Enterobacteriaceae (7-9). However, almost all of the CTX-M-9 group of enzymes were found in E. coli in the present study, and some of them were suggested to be CTX-M-14. Precise analysis of the genetic environments mediating the blaCTX-M-9 group of genes among these strains as well as their genome profiles would explain the presence of CTX-M-producing pandemic strains inJapan.
In conclusion, the aim of the present study was to make a rough estimate of the current status of CTX-M-type β-lactamases produced by nosocomial gram-negative bacilli isolated from Japanese medical facilities. The findings obtained imply that various plasmid-mediated genetic determinants for CTX-M-type β-lactamases have already been disseminated in Japanese clinical environments. Since CTX-M-2 was also identified in livestock (24), we must take special precautions against the further proliferation of gram-negative bacterial strains that harbor plasmids carrying genes for CTX-M-type β-lactamases, together with the other classes of plasmid-mediated β-lactamases, such as CMY-type cephamycinases and MBLs.
Acknowledgments
We thank all medical institutions for submitting bacterial strains to the national reference laboratory for performance of this study.
This work was mainly supported by two grants (grants H15-Shinko-9 and H15-Shinko-10) from the Ministry of Health, Labor and Welfare of Japan. PCR typing of bla genes was supported by grants (grants 1017221 and 13770141) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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