Abstract
Twenty-seven well-characterized metallo-β-lactamase (MBL)-producing Pseudomonas strains from two distantly located hospitals were analyzed. The results revealed specific features defining the multilevel epidemiology of strains from each hospital in terms of species, clonality, predominance of high-risk clones, composition/diversity of integrons, and linkages of Tn402-related structures. Therefore, despite the global trends driving the epidemiology of MBL-producing Pseudomonas spp., the presence of local features has to be considered in order to understand this threat and implement proper control strategies.
TEXT
The growing incidence of horizontally acquired resistance in Pseudomonas aeruginosa and other related nonfermenters (such as Pseudomonas putida), added to its extraordinary capacity to acquire resistance through chromosomal mutations, dramatically limits our arsenal for the treatment of nosocomial infections (1, 2). In this regard, the transferable class B carbapenemases, also known as metallo-β-lactamases (MBLs), are of particular concern. While multiple reports denote a growing prevalence and diversity of such enzymes worldwide, linked to class 1 integrons and particular clones in certain areas (3, 4), there is still scarce information on the transferable elements responsible for their dissemination and whether these elements are different or similar in different geographical locations. Thus, in order to define common or specific epidemiological traits, we performed a multilevel comparison (clones, enzymes, integrons, and particularly transferable elements) of the epidemiology of MBL-positive Pseudomonas spp. from two distantly located (approximately 300 km) Spanish hospitals.
For this purpose, a total of 27 isolates from two previously established collections of MBL-positive Pseudomonas strains detected between June 2008 and June 2010 in the Hospital Clínico Universitario Lozano Blesa (HLB) (848 beds) (Zaragoza, Spain), and Hospital 12 de Octubre (H12O) (1,300 beds) (Madrid, Spain) were used (5, 6). The H12O collection included one isolate representing each Pseudomonas sp. pulsed-field gel electrophoresis (PFGE) clone proved to harbor an MBL during the study period, 5 P. putida and 6 P. aeruginosa clones. Additionally, due to the extraordinary epidemic spread (104 patients between 2007 and 2010) of an additional P. aeruginosa clone (the previously described clone B), 4 representative samples of it were also studied in order to find potential differences in their acquired resistance determinants (5, 7). Hence, the H12O collection constituted a total of 10 P. aeruginosa samples. Regarding the isolates collected from HLB, no MBL-positive isolates of Pseudomonas species different from P. aeruginosa were detected, but a high number of different clones was found (up to 30), from which 12 strains of representative pulsotypes were selected (6). The clinical data, resistance profiles, and clonal relatedness (PFGE and multilocus sequence typing [MLST] analysis) of both collections are shown in Table 1. Most of the 27 cases (89%) were considered infections, presenting a crude 30-day mortality of 15%. As expected, the most frequently involved hospital department was the intensive care unit (ICU) (59%), and the acquisition of the infection occurred in patients on average 55.8 days after admission, without significant differences between the two participating hospitals (Table 1). Twenty-five of the 27 strains produced VIM-2 (one was additionally positive for VIM-1), while the remaining two strains produced VIM-1 or IMP-22 (Table 1). MBLs were frequently linked to globally spread high-risk clones (8–14) in both hospitals with different distributions. Sequence type 111 (ST111) and ST244 were detected only among H120 strains, whereas ST235 was detected only in the HLB strains. Moreover, MLST analysis indicated distinct wide dissemination of ST175 in the H12O hospital and ST235 in HLB (represented by 8 different pulsotypes). In this sense, the low number of different STs harboring MBLs (just three) among the HLB isolates was remarkable in contrast with the relatively high number of STs (six) in H12O. Likewise, polyclonal MBL-positive P. putida strains, found to act as environmental reservoirs of genetic elements harboring these resistance determinants (8, 13), were also found only among H12O isolates.
TABLE 1.
Clinical data of patients and molecular epidemiology and antimicrobial susceptibility of all the strains included in this study
| Strain code | Species | Hospitala (ward) | Patient gender (age [yr]) | Underlying pathologyb | Sample (C/I)c | Date of isolation (day/mo/yr [days of admission before isolation]) | Crude 30-day mortality | PFGE | ST | MBL | Resistance patternd |
|---|---|---|---|---|---|---|---|---|---|---|---|
| W335 | P. aeruginosa | HLB (ICU) | M (64) | Subarachnoid hemorrhage | Urine (I) | 03/07/08 (37) | No | HLB-3 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, CIP |
| W340 | P. aeruginosa | HLB (ICU) | M (49) | Subdural hematoma | Bronchial aspirate (I) | 04/07/08 (38) | No | HLB-11 | 973 | VIM-2 | IMP, MER, FEP, GEN, TOB, AMK, CIP |
| W336 | P. aeruginosa | HLB (ICU) | F (77) | Eventration | Bronchial aspirate (I) | 05/07/08 (115) | Yes | HLB-8 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, CIP |
| W343 | P. aeruginosa | HLB (ICU) | F (21) | Viral meningoencephalitis | Urine (I) | 16/07/08 (76) | No | HLB-6 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, CIP |
| W368 | P. aeruginosa | HLB (ICU) | F (45) | Community pneumonia with septic shock | Urine (I) | 20/02/09 (45) | No | HLB-12 | 973 | VIM-2 | IMP, MER, CAZ, GEN, TOB, AMK, CIP |
| W378 | P. aeruginosa | HLB (ICU) | M (45) | TBC, HCV, renal insufficiency | Surgical wound (I) | 07/05/09 (18) | No | HLB-7 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, CIP |
| Ps24 | P. aeruginosa | HLB (ICU) | F (65) | Subarachnoid hemorrhage | Bronchial aspirate (I) | 22/07/09 (13) | No | HLB-10 | 973 | VIM-2 | IMP, MER, CAZ, FEP, GEN, TOB, AMK, CIP |
| Ps27 | P. aeruginosa | HLB (ICU) | M (57) | Goodpasture syndrome with lung hemorrhage | Bronchial aspirate (I) | 17/08/09 (10) | Yes | HLB-4 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, CIP |
| Ps56 | P. aeruginosa | HLB (HOS) | M (77) | Chronic prostatitis | Sputum (I) | 18/12/09 (123) | No | HLB-9 | 175 | VIM-2 | IMP, CAZ, ATM, GEN, TOB, AMK, CIP |
| Ps12 | P. aeruginosa | HLB (ICU) | F (50) | Cardiogenic shock | Pharynx exudate (I) | 18/12/09 (16) | Yes | HLB-1 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, AMK, CIP |
| Ps32 | P. aeruginosa | HLB (ICU) | F (62) | Septic shock | Urine (I) | 23/03/10 (41) | No | HLB-2 | 235 | VIM-2 | IMP, MER, CAZ, FEP, GEN, TOB, AMK, CIP |
| Ps60 | P. aeruginosa | HLB (ICU) | F (52) | Carcinoma of ampulla | Urine (I) | 28/05/10 (31) | No | HLB-5 | 235 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, CIP |
| 12O4 | P. aeruginosa | H12O (DS) | M (74) | BOOP | Sputum (C) | 07/08/08 (14) | No | 12O-B | 175 | VIM-2 | IMP, CAZ, FEP, PTZ, GEN, TOB, CIP |
| 12O5 | P. aeruginosa | H12O (PNE) | F (36) | Epilepsy | Urine (I) | 16/10/08 (122) | No | 12O-F | 111 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, AMK, CIP |
| 12O9 | P. aeruginosa | H12O (ICU) | M (48) | Chronic hepatic pathology | Bile (I) | 22/12/08 (40) | No | 12O-C | 450 | VIM-2 | IMP, CAZ, FEP, PTZ, ATM, GEN, TOB, AMK, CIP |
| 12O10 | P. aeruginosa | H12O (DS) | M (79) | Chronic respiratory failure | Sputum (I) | 29/12/08 (15) | No | 12O-G | 244 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, AMK, CIP |
| 12O11 | P. putida | H12O (IM) | F (51) | Cardiovascular pathology | Inguinal exudate (C) | 13/11/08 (5) | No | 12O-H | - | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, AMK, CIP |
| 12O12 | P. aeruginosa | H12O (ICU) | M (65) | Cardiovascular pathology | Blood culture (I) | 26/02/09 (30) | No | 12O-I | 155 | VIM-1 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, AMK, CIP |
| 12O13 | P. aeruginosa | H12O (ICU) | F (53) | Obstruction intestinal syndrome | Urine (I) | 03/04/09 (82) | No | 12O-J | 244 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, CIP |
| 12O14 | P. putida | H12O (DAM) | F (82) | Actinic enteritis | Urine (I) | 27/04/09 (160) | No | 12O-K | - | VIM-2 | IMP, MER, CAZ, PTZ, GEN, TOB, CIP |
| 12O19 | P. putida | H12O (GS) | M (77) | Rectal adenocarcinoma | Blood culture (I) | 01/10/09 (28) | No | 12O-L | - | VIM-2 | IMP, MER, CAZ, PTZ, GEN, TOB, CIP |
| 12O21 | P. aeruginosa | H12O (IM) | M (58) | Solid tumor | Blood culture (I) | 10/02/10 (324) | No | 12O-B | 175 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, CIP |
| 12O23 | P. aeruginosa | H12O (ICU) | M (58) | Abdominal perforation | Bile (I) | 02/03/10 (10) | Yes | 12O-B | 175 | IMP-22 | IMP, MER, CAZ, FEP, GEN, TOB, CIP |
| 12O25 | P. putida | H12O (ICU) | M (71) | Gastric carcinoma | Urine (I) | 11/05/10 (33) | No | 12O-M | - | VIM-1 and VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, CIP |
| 12O26 | P. aeruginosa | H12O (ONC) | F (70) | BOOP | Sputum (I) | 10/05/10 (42) | No | 12O-B | 175 | VIM-2 | IMP, MER, CAZ, FEP, PTZ, GEN, TOB, CIP |
| 12O27 | P. putida | H12O (PNE) | M (74) | Deep vein thrombosis | Urine (C) | 12/05/10 (14) | No | 12O-N | - | VIM-2 | IMP, MER, CAZ, FEP, PTZ, ATM, GEN, TOB, AMK, CIP |
| 12O28 | P. aeruginosa | H12O (IM) | F (89) | Cholangiocarcinoma | Catheter (I) | 28/05/10 (26) | No | 12O-O | 253 | VIM-2 | IMP, MER, PTZ, GEN, TOB, CIP |
HLB, Hospital Clínico Universitario Lozano Blesa; H12O, Hospital 12 de Octubre; ICU, intensive care unit; HOS, general hospitalization; DS, digestive surgery; PNE, pneumology; IM, internal medicine; DAM, digestive apparatus medicine; GS, general surgery; ONC, oncology.
TBC, tuberculosis; HCV, hepatitis C virus; BOOP, bronchiolitis obliterans organizing pneumonia.
C, colonization; I, infection.
The displayed antibiotics are those to which each strain showed clinical resistance, following the 2013 version 3.1 EUCAST breakpoints (www.eucast.org/clinical_breakpoints/). IMP, imipenem; MER, meropenem; CAZ, ceftazidime; FEP, cefepime; PTZ, piperacillin-tazobactam; ATM, aztreonam; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin.
The composition of the MBL-carrying integrons was analyzed through PCR amplification and sequencing following previously described protocols (8, 15, 16), and results are shown in Fig. 1. Those combinations of cassettes not previously described were uploaded on the Integrall database (http://integrall.bio.ua.pt/), and the provided nomenclature is also shown in Fig. 1. Interestingly, most of the HLB blaVIM-2 integrons were quite uniform in their simplicity, given that in all the strains but two (PA-HLB-Ps12 and PA-HLB-W378), blaVIM-2 was the unique cassette detected in the variable region (In56). This fact suggests the potential recent and contemporary acquisition of the VIM-2-carrying integrons in most of the strains, which would not have had enough time to accumulate new cassettes. On the other hand, the sequencing of the PA-HLB-W378 strain integron revealed a partial structure, containing ISPa34, which has been previously described in another Spanish hospital (17). Interestingly, exactly the same complete integron from the latter work (codifying two copies of blaVIM-2 among other genes) was found in the strain PA-HLB-W336, which additionally harbored another class 1 integron with only blaVIM-2. The integrons of H12O strains were much more variable, showing different combinations of MBLs and aminoglycoside-modifying enzymes, even with a strain (PP-H12O-25) harboring blaVIM-1 and blaVIM-2 codified in two different integrons. Moreover, two P. putida strains from H12O harbored a new aminoglycoside-modifying enzyme gene variant, designated aac(6′)-49, presumably with acetyltransferase activity, given its homology with other aminoglycoside 6′-N-acetyltransferases (53 to 55% amino acid identities). Five of the 11 MBL-harboring integron structures detected were novel; In896, In902, In903, In908, and In927 were most detected in H12O (Fig. 1).
FIG 1.
Composition of the MBL-carrying integrons and their genetic context. The gray boxes/arrows represent the genes belonging to Tn402-related structures carrying the integrons. The thick black lines interrupted by two diagonals represent the areas that could not be amplified by PCR. The black oval dots represent the intergenic DNA. The integrons' names and their cassettes were obtained in the Integrall database (http://integrall.bio.ua.pt/). The dashes in the column labeled “Integron name” indicate an incomplete integron structure; the Integrall database (http://integrall.bio.ua.pt/) does not provide In- names for partial integrons. Abbreviations in column 2: PA, Pseudomonas aeruginosa; PP, Pseudomonas putida; ST, sequence type; HLB, Hospital Clínico Universitario Lozano Blesa; H12O, Hospital 12 de Octubre. Location “C” represents “chromosomal.”
Most of the studies dealing with MBL-carrying integrons focus on their cassette composition, but fewer studies have investigated the genetic context where the integrons are inserted, in order to understand the potential mechanisms of dissemination and particularities of their origin. To gain insights into these matters, PCRs using previously described overlapping primers for the amplification of tni genes (tniQ, tniC, tniB, and tniA, belonging to Tn402-like structures), often located downstream of the 3′-conserved segment (3′CS) of class 1 integrons, and subsequent sequencing were performed (8, 15, 18, 19). The resulting sequences were then compared with those on GenBank, and the obtained data are shown in Fig. 1. The location of MBL integrons within transposon structures deriving from Tn402, a widely reported pattern for the class 1 integrons, was very frequent (19 of the 27 studied strains) (Fig. 1). However, the structures of integrons lacking the whole 3′CS (qacE1, sul1, and ORF5) and showing a complete tni module instead (tniC, tniQ, tniB, and tniA), which have already been described in our country and elsewhere (15, 18), were not detected in any of the strains from the two hospitals. These particular integron-tni complete module structures have been proposed to be still-functional transposable elements, in contrast to those integrons with complete 3′CS but a truncated tni module, which are thus transposition-defective transposon derivatives that would need to be embedded in additional larger transposons to be mobilized (18–20). Precisely these truncated variants were those most usual in our work (19 of the total 27 strains), with a high degree of homology in the tniBΔ and tniA nucleotide sequences (more than 97% among all the strains). The presence of particular Tn402-like variants, with insertion sequences interrupting tniB, was exceptional. Only the PA-HLB-Ps56 strain showed an IS1326 between ORF5 and tniB. However, the widely described presence of IS1353 upstream of IS1326 was not found in this strain, constituting a rare variant of Tn402-like structure, probably indicating a very particular origin proceeding from the described In0 (19, 21, 22). Another particular structure, containing a different specific deletion site for tniB, previously described as tniBΔ3 (19), was found only in one strain, PA-H12O-12, also suggesting a very particular and differential origin for this integron (Fig. 1). In contrast, in most cases (17 strains), the PCRs for partial tniB (disrupted in a common site yielding a particular tniB fragment previously denominated as tniBΔ1) and tniA genes provided positive results (Fig. 1). Therefore, the sharing of specific transposon structures, different from those of integrons with the complete tni module, as has been shown for the U.S., Russian, and Indian class 1 integrons (22, 23), suggests a single ancestral origin for most of the MBL-harboring genetic elements described in this work, detected among strains from both participating hospitals and among both P. aeruginosa and P. putida isolates. For 8 of the strains, neither tniA nor tniB could be amplified using different combinations of primers, suggesting the presence of alternative, not yet described genetic elements carrying the integrons of these strains that need to be explored in future studies. This alternative structure was almost exclusively detected in HLB strains, further supporting the concept that each health center shows particular and not generalizable epidemiologic features, including the origin of the dominant MBL integrons.
Finally, regarding the potential plasmid/chromosomal location of the different MBLs, all the attempts to transfer the plasmid DNA (obtained using the Ultraclean Plasmid Prep kit; Mo-Bio) to the PAO1 reference strain through electroporation/conjugation yielded negative results. Furthermore, the Southern blot hybridization following described protocols (15) using the North2South complete biotin random primer labeling and detection kit (Thermo Scientific), over the I-CeuI/S1 nuclease-digested genomes, suggested the chromosomal location of MBL genes in all the strains, given that the respective MBL gene probes hybridized with bands which also hybridized with the rRNA gene probe (data not shown). This finding is supported by several previous studies that have also shown that the majority of MBL genes are chromosomally located in P. aeruginosa, probably due to a poor replication of plasmids within this species, and the necessity of a chromosome integration of the genes (24, 25). Moreover, chromosomal location was also observed in all P. putida strains analyzed. This result contrasts with those of a different hospital in Spain for which in the vast majority of MBL-producing P. putida strains plasmid locations were demonstrated (8).
In conclusion, despite the uniform dominance of VIM-2 production, our results indicate the presence of specific features defining the multilevel epidemiology of MBL-producing Pseudomonas spp. in each of the two participating hospitals, in terms of Pseudomonas species, clonal diversity, predominance of particular high-risk clones, and composition and diversity of integrons and their linkage to Tn402-related structures. Therefore, despite clear global trends driving the epidemiology of MBL-producing Pseudomonas spp., the presence of specific local features also needs to be considered for understanding this growing threat and implementing optimal control strategies. In light of our findings, the measures to be implemented at the local level should likely include the detection and control of the so-called high-risk clones, even if they have not yet acquired MBL determinants, and the search for environmental reservoirs of MBL-producing genetic elements before they are successfully transferred to those high-risk clones.
ACKNOWLEDGMENTS
This work was supported by the Ministerio de Economía y Competitividad of Spain and the Instituto de Salud Carlos III, through the Spanish Network for the Research in Infectious Diseases (no. RD06/0008 and RD12/0015), the projects FIS PI12/01276 and SAF2012-35474, the Direcció General d′Universitats, Recerca i Transferència del Coneixement del Govern de les IllesBalears, and Fondo Europeo de Desarrollo Regional (FEDER). During the experimental work for this study, V. Estepa had a predoctoral fellowship from the University of La Rioja, Spain.
Footnotes
Published ahead of print 3 February 2014
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