Abstract
A 6-month, PCR-based, island-wide hospital surveillance study of beta-lactam resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii was conducted in Puerto Rico. Of 10,507 isolates, 1,239 (12%) unique, multi-beta-lactam-resistant isolates from all geographical regions were identified. The KPC gene was detected in 61 E. coli, 333 K. pneumoniae, 99 P. aeruginosa, and 41 A. baumannii isolates, indicating the widespread dissemination of the KPC gene in clinically significant nosocomial isolates.
TEXT
During the past decades, the emergence of multi-beta-lactam-resistant (MβLR) Gram-negative bacilli has become an important clinical problem associated with increases in mortality rates and the length and cost of hospital stays. Acquired broad-spectrum beta lactamases have been identified with increasing frequency in clinical isolates of Gram-negative bacilli (2, 7). KPC (Klebsiella pneumoniae carbapenemase) belongs to the Ambler class A, Bush subgroup 2f, serine-based carbapenemases, which are active against all beta-lactams, including the carbapenems (9). Ten KPC variants (KPC-2 to -11) are currently known. The KPC enzyme has been detected worldwide in Enterobacteriaceae and recently in Pseudomonas aeruginosa isolates from Colombia, Puerto Rico, Trinidad and Tobago, and the United States and in Acinetobacter baumannii in Puerto Rico (1, 8, 11, 13). The KPC gene has been found associated with the plasmid-borne transposon Tn4401, which may be responsible for its rapid dissemination (4, 6).
Previous studies conducted in Puerto Rico have detected a significant number of KPC-positive Gram-negative bacilli in Puerto Rico Medical Center hospitals (10–12, 14). Pulsed-field gel electrophoresis of these isolates showed both clonally related and unrelated isolates. In September of 2008, the first outbreak caused by a KPC-positive K. pneumoniae strain was identified in a hospital located in the southern region of Puerto Rico (5). The aim of this study was to perform an island-wide surveillance study to identify and determine the geographical distribution of KPC-positive nosocomial Gram-negative bacilli.
A PCR-based surveillance study of beta-lactam resistance was conducted during a 6-month period (January to June 2009) in 17 hospitals across the island. Participating hospitals provided all unique, consecutive, MβLR Escherichia coli, K. pneumoniae, P. aeruginosa, and A. baumannii isolates, together with the corresponding susceptibility reports and basic epidemiologic information. Multi-beta-lactam resistance was defined as resistance to any of the carbapenems and/or two or more of the following antibiotics: ceftriaxone, cefotaxime, ceftazidime, cefepime, aztreonam, and piperacillin-tazobactam. The KPC PCR assay was performed utilizing primers and conditions as previously described (14). No attempts were made to evaluate patients' therapies or clinical outcomes. Statistical analysis was performed utilizing the two-tailed Fisher exact test. A P value of ≤0.05 was considered statistically significant.
Table 1 shows the total number of isolates together with the MβLR and KPC-positive organisms identified during the study period. Using the monthly bacteriology laboratory reports submitted by the participating hospitals, 10,507 E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii clinical isolates were identified. This, however, represents an overestimation due to the collection of multiple identical isolates from the same patients. A total of 1,239 unique, consecutive MβLR isolates were identified, representing 12% of the 10,507 isolates. The KPC gene was detected in 5% (534/10,507) of the total and 43% (534/1,239) of the MβLR isolates. The distribution of the KPC gene among the MβLR isolates was as follows: E. coli, 28% (61/219); K. pneumoniae, 73% (333/457); P. aeruginosa, 36% (99/272); A. baumannii, 14% (41/291). There was a statistically significantly higher number of KPC-positive Klebsiella pneumoniae isolates than of each of the other three organisms (P ≤ 0.05) in all regions.
Table 1.
Numbers of KPC-positive E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii isolates among the total number of isolates and the multi-beta-lactam-resistant isolates
| Organism | No. of isolates |
No. of KPC producers/total (%) |
|||
|---|---|---|---|---|---|
| Total | MβLR | KPC producers | All isolates | MβLR isolates | |
| E. coli | 4,329 | 219 | 61 | 61/4,329 (1.4) | 61/219 (33) |
| K. pneumoniaea | 2,805 | 457 | 333 | 333/2,805 (12) | 333/457 (73) |
| P. aeruginosa | 2,415 | 272 | 99 | 99/2,415 (4.1) | 99/272 (44) |
| A. baumannii | 958 | 291 | 41 | 41/958 (4.3) | 41/291 (14) |
| Total | 10,507 | 1,239 | 534 | 534/10,507 (5) | 534/1,239 (43) |
P ≤ 0.05.
Table 2 shows the total number of hospitals, beds, and KPC-positive isolates by geographic region. The metropolitan region had the highest number of hospital beds. Comparison of the numbers of KPC-positive isolates of these organisms by geographic region showed that the number of E. coli isolates was significantly higher in the southern and Puerto Rico Medical Center regions (P ≤ 0.05) and that of P. aeruginosa was significantly higher in the Puerto Rico Medical Center and western regions (P ≤ 0.05), while K. pneumoniae and A. baumannii were equally distributed among all areas.
Table 2.
Total numbers of hospitals, beds, and KPC-positive isolates by geographic region
| Geographic region | Total no. (%) of: |
No. of KPC-positive isolates/total (%) |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Hospitals | Beds | Isolates | MβLR isolates | KPC producers | E. coli | K. pneumoniaea | P. aeruginosa | A. baumannii | Overall | |
| Puerto Rico Medical Center | 6 | 858 | 2,472 | 354 | 180 | 19/32 (59)a | 101/122 (83) | 41/67 (61)a | 19/133 (14) | 180/354 (51) |
| Metropolitan | 3 | 906 | 2,371 | 196 | 72 | 8/52 (15) | 39/59 (66) | 18/46 (39) | 7/39 (18) | 72/196 (37) |
| North | 2 | 439 | 1,706 | 259 | 87 | 15/73 (21) | 65/94 (69) | 3/56 (5) | 4/36 (11) | 87/259 (34) |
| West | 2 | 324 | 1,316 | 95 | 43 | 1/13 (8) | 26/38 (68) | 15/27 (56)a | 1/17 (6) | 43/95 (45) |
| South | 2 | 593 | 1,299 | 176 | 86 | 15/35 (43)a | 55/75 (73) | 13/48 (27) | 3/18 (17) | 86/176 (49) |
| Central/East | 2 | 497 | 1,343 | 159 | 66 | 3/14 (21) | 47/69 (68) | 9/28 (32) | 7/48 (15) | 66/159 (42) |
| Total | 17 | 3,617 | 10,507 | 1,239/10,507 (12) | 534/1,239 (43) | 61/219 (28) | 333/457(73) | 99/272 (36) | 41/291 (14) | 534/1239 (43) |
P ≤ 0.05.
Table 3 shows the baseline epidemiological information of the KPC-positive isolates. KPC-positive isolates were similarly distributed between cultures obtained from male and female patients. Samples obtained from the respiratory and urinary tracts yielded significantly higher numbers of such organisms than those obtained from other anatomical sites (P ≤ 0.05). The distribution of the KPC-positive isolates by hospital area revealed that 167 were identified in the intensive care unit (ICU) and 349 in the general wards. The reason(s) for this difference is not clear from our data; it could suggest simply a higher number of specimens obtained from the general wards, the transfer of infected or colonized patients from the ICU to the general wards, hospitalization of patients already colonized with KPC-positive isolates, and/or that the organisms are not confined to a specific hospital area. Table 4 shows the antibiotic susceptibilities of the KPC-positive isolates to selected agents. The imipenem susceptibility breakpoints were reported as ≤4 μg/ml since the samples were collected prior to the June 2010 Clinical and Laboratory Standards Institute carbapenem susceptibility breakpoint changes. With the exception of the susceptibility of E. coli to imipenem (83%), the antimicrobial activity of the beta- and non-beta-lactam antibiotics was marginal to very poor. Unfortunately, susceptibility to polymyxins and tigecycline was not reported. Phenotypic detection of extended-spectrum beta-lactamases (ESBLs) was observed in 24% and 62% of the KPC-positive K. pneumoniae and E. coli isolates, respectively. These results are in agreement with the recent literature reports and suggest the presence of multiple different mechanisms of antibiotic resistance in these isolates (2, 3, 7).
Table 3.
Baseline clinical information on KPC-positive E. coli, K. pneumoniae K. pneumoniae, P. aeruginosa, and A. baumannii isolates
| Organism | Total no. of KPC-positive isolates | No. of isolates from patients of following gender: |
No. of isolates from following hospital unit: |
No. of isolates from following anatomical site: |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Fa | Mb | NRc | ICU | General ward | NR | RTd,h | UTe,h | SSTf | Blood | Misc.g | NR | ||
| E. coli | 61 | 26 | 35 | 0 | 13 | 44 | 4 | 9 | 23 | 12 | 10 | 7 | 0 |
| K. pneumoniae | 333 | 162 | 167 | 4 | 108 | 212 | 13 | 90 | 93 | 57 | 49 | 42 | 2 |
| P. aeruginosa | 99 | 46 | 50 | 3 | 28 | 71 | 0 | 35 | 22 | 18 | 10 | 13 | 1 |
| A. baumannii | 41 | 18 | 22 | 1 | 18 | 22 | 1 | 16 | 4 | 7 | 8 | 5 | 1 |
| Total | 534 | 252 | 274 | 8 | 167 | 349 | 18 | 150 | 142 | 94 | 77 | 67 | 4 |
F, female.
M, male.
NR, not reported.
RT, respiratory tract.
UT, urinary tract.
SST, skin and soft tissue.
Misc., miscellaneous.
P ≤ 0.05.
Table 4.
Total numbers and percentages of isolates susceptible to selected antibioticsa
| Organism | Total no. of KPC producers | No. of isolates susceptible/total (%) |
||||||
|---|---|---|---|---|---|---|---|---|
| Ceftriaxone | Cefepime | Imipenem | Piperacillin-tazobactam | Amikacin | Ciprofloxacin | ESBL | ||
| K. pneumoniae | 333 | 21/288 (7) | 31/300 (10) | 89/316 (28) | 7/299 (2) | 114/330 (35) | 46/332 (14) | 80/332 (24) |
| E. coli | 61 | 10/52 (19) | 10/51 (20) | 44/53 (83) | 24/52 (46) | 39/60 (65) | 10/60 (17) | 37/60 (62) |
| P. aeruginosa | 99 | 2/92 (2) | 6/98 (6) | 6/95 (6) | 16/98 (16) | 62/95 (59) | 7/98 (7) | NDb |
| A. baumannii | 41 | 0/40 (0) | 0/40 (0) | 11/33 (33) | 2/34 (6) | 4/41 (10) | 0/41 (0) | ND |
Data, including those on ESBL detection, are from the participating hospitals' antimicrobial susceptibility reports. The susceptibility breakpoints (μg/ml) for all isolates are as follows: ≤8, cefepime; ≤4, imipenem; ≤16, amikacin; ≤1, ciprofloxacin. The susceptibility breakpoints of ceftriaxone are ≤1 μg/ml for Enterobacteriaceae and ≤8 μg/ml for P. aeruginosa and A. baumannii. The susceptibility breakpoints (μg/ml) of ceftriaxone are ≤1 and ≤8 for the Enterobacteriaceae P. aeruginosa and A. baumannii. The susceptibility breakpoints (μg/ml) of piperacillin-tazobactam are ≤64 and 4 for P. aeruginosa and ≤16 and 4 for A. baumannii.
ND, not done.
This surveillance study clearly demonstrated that the KPC gene has readily spread among important nosocomial pathogens in Puerto Rico. The reasons for this dissemination are not clear from our results; however, it can be speculated that multiple social and microbiological factors may be at play, such as: the small size of the island (3,435 square miles) with a high population density of 1,158.5 inhabitants per square mile (2009 estimate); the ease of ground transportation that facilitates the movement of patients to different hospitals; constant air travel between Puerto Rico and the continental United States and other countries; the extensive use of broad-spectrum antibiotics due to the high number of ESBL-positive isolates; antibiotic misuse; lax infection control practices; and/or the horizontal transmission of the KPC and other antibiotic resistance genes. This study clearly emphasizes the importance of prompt recognition of these isolates and the establishment of proper therapeutic and infection control measures to reduce the spread of these organisms among patients and within hospitals.
Acknowledgments
This work was supported by Janssen Ortho-McNeil (Johnson & Johnson), Inc.; Merck Sharp and Dohme, Inc.; Pfizer, Inc.; the Puerto Rico Department of Health; and NCRR/NIH-RCMI award G12RR03051.
We acknowledge the following physician members of the Puerto Rico Antibiotic Resistance Study Group: Miguel Colón, Osvaldo Laboy, Carlos F. León, Agripino Lugo, Vanessa Olivo, Diana M. Otero, Ramón Ramírez Ronda, Jorge L. Santana, María I. Santé, and Nilda Zapata. We thank the participating hospitals' bacteriology laboratory personnel for collecting the isolates and epidemiological information, in particular, Carmen Báez, Myriam Corazón, Madeline Cruz, Leyda E. Echevarría, Maria Maldonado, Aixa Martínez, María Matos, Miriam Nistal, Nereida Santiago, Linnette Santos, Abigail Torres, and Nayda Vázquez. We are grateful for the support of Ada M. Cortez, Enid J. García, and Johnny Rullán from the office of Epidemiology and Research, Puerto Rico Department of Health. We appreciate the dedicated technical assistance of Caleb Fernández, graduate student Teresa Martínez, and the undergraduate students of our laboratory. We also thank Wieslaw J. Kozek and Philip C. Specht for reviewing the manuscript.
Footnotes
Published ahead of print on 28 March 2011.
REFERENCES
- 1. Akpaka P. E., et al. 2009. Emergence of KPC-producing Pseudomonas aeruginosa in Trinidad and Tobago. J. Clin. Microbiol. 47:2670–2671 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Bush K. 2010. Alarming beta-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr. Opin. Microbiol. 13:558–564 [DOI] [PubMed] [Google Scholar]
- 3. Castanheira M., Sader H. S., Jones R. N. 2010. Antimicrobial susceptibility patterns of KPC-producing or CTX-M-producing Enterobacteriaceae. Microb. Drug Resist. 16:61–65 [DOI] [PubMed] [Google Scholar]
- 4. Curiao T., et al. 2010. Emergence of bla KPC-3-Tn4401a associated with a pKPN3/4-like plasmid within ST384 and ST388 Klebsiella pneumoniae clones in Spain. J. Antimicrob. Chemother. 65:1608–1614 [DOI] [PubMed] [Google Scholar]
- 5. Gregory C. J., et al. 2010. Outbreak of carbapenem-resistant Klebsiella pneumoniae in Puerto Rico associated with a novel carbapenemase variant. Infect. Control Hosp. Epidemiol. 31:476–484 [DOI] [PubMed] [Google Scholar]
- 6. Naas T., et al. 2008. Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene. Antimicrob. Agents Chemother. 52:1257–1263 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Nordmann P., Cuzon G., Naas T. 2009. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect. Dis. 9:228–236 [DOI] [PubMed] [Google Scholar]
- 8. Poirel L., Nordmann P., Lagrutta E., Cleary T., Munoz-Price L. S. 2010. Emergence of KPC-producing Pseudomonas aeruginosa in the United States. Antimicrob. Agents Chemother. 54:3072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Queenan A. M., Bush K. 2007. Carbapenemases: the versatile beta-lactamases. Clin. Microbiol. Rev. 20:440–458 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Robledo I. E., et al. 2008. Dissemination and molecular epidemiology of KPC producing K. pneumoniae (Kp) collected in the Puerto Rico Medical Center Hospitals (PRMCH) during 2003-04, abstr. C2-3734. Abstr. 48th Intersci. Conf. Antimicrob. Agents Chemother., Washington, DC, 25 and 26 October, 2008 [Google Scholar]
- 11. Robledo I. E., et al. 2010. Detection of KPC in Acinetobacter spp. in Puerto Rico. Antimicrob. Agents Chemother. 54:1354–1357 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Vázquez G. J., et al. 2003. A comparison of the antimicrobial resistance patterns of gram-negative bacilli isolated from community-private and university-affiliated hospitals from Puerto Rico. P. R. Health Sci. J. 22:265–271 [PubMed] [Google Scholar]
- 13. Villegas M. V., et al. 2007. First identification of Pseudomonas aeruginosa isolates producing a KPC-type carbapenem-hydrolyzing beta-lactamase. Antimicrob. Agents Chemother. 51:1553–1555 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Wolter D. J., et al. 2009. Surveillance of carbapenem-resistant Pseudomonas aeruginosa isolates from Puerto Rican Medical Center Hospitals: dissemination of KPC and IMP-18 beta-lactamases. Antimicrob. Agents Chemother. 53:1660–1664 [DOI] [PMC free article] [PubMed] [Google Scholar]