Research in Microbiology 165 (2014) 852e856 www.elsevier.com/locate/resmic
Brief note
Establishment and multi drug resistance evolution of ST235 Pseudomonas aeruginosa strains in the intensive care unit of a Colombian hospital Elena Martinez a,*, Javier Escobar Perez b, Francisco Buelvas c, Catalina Tovar c, Natasha Vanegas a,b, H.W. Stokes a a
The ithree Institute, University of Technology, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia b Laboratorio de Genetica Molecular Bacteriana, Universidad El Bosque, Bogota, Colombia c Grupo de Resistencia Bacteriana y Enfermedades Tropicales, Universidad del Sinu´, Monteria-Cordoba, Colombia Received 14 May 2014; accepted 21 October 2014 Available online 1 November 2014
Abstract Drug resistant Pseudomonas aeruginosa represents a therapeutic challenge. To assess the diversity of P. aeruginosa antibiotic resistant variants, isolates were recovered from hospital patients in Colombia. Thirty of 60 isolates contained class 1 integrons and five were of Sequence Type ST235 having appeared in a single intensive care unit. All five possessed an unusual integron but showed differences in gene cassette content and the presence/absence of insertion sequence IS26. This showed that differences can arise rapidly, even within a single ICU. Also, the emergence of IS26 in P. aeruginosa is contributing to the evolution of resistance in this bacterium. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Class 1 integron; Pseudomonas aeruginosa ST235; IS26; Antibiotic resistance; Hospital acquired infections
1. Introduction Pseudomonas aeruginosa is one of the ESKAPE group of pathogens [1] and is a bacterium that is increasingly recalcitrant to antibiotic therapy. P. aeruginosa is also a significant opportunistic pathogen that manifests in a number of infection contexts most notably in relation to burns victims, cystic fibrosis sufferers and intensive care unit (ICU) patients. One reason P. aeruginosa is increasingly difficult to treat with antibiotics is that it possesses regions of great genomic plasticity indicating substantial amounts of lateral gene transfer (LGT). This LGT can be facilitated by several mobilizing elements including plasmids, transposons and integrons. Many * Corresponding author. E-mail addresses: [email protected] (E. Martinez), [email protected] (J.E. Perez), [email protected] (F. Buelvas), [email protected] (C. Tovar), [email protected] (N. Vanegas), [email protected] (H.W. Stokes).
of these elements and linked genes frequently cluster in chromosomally located genomic islands. These strain specific regions can comprise hundreds of kb in pathogenic strains. Notwithstanding the high level of genome variability it is evident that particular sequence types (ST), as defined by multi locus sequence typing (MLST), appear to be particularly infectious and virulent as measured by their global spread in clinical settings. One of the best examples of this is ST235 which has become broadly established across Europe [2,3]. The clone is also reported in other parts of the world [4] including South America [5] although less commonly. ST235 isolates are commonly associated with the dissemination of particular resistance genes. This is especially true of genes conferring resistance to clinically important b-lactam antibiotics. For example VIM producers have become prevalent in parts of Europe as a consequence of the spread of ST235 clones [3,6]. ST235 strains should not be considered identical however since resistance gene content can vary greatly as the plasticity of P. aeruginosa has allowed on going
http://dx.doi.org/10.1016/j.resmic.2014.10.011 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
E. Martinez et al. / Research in Microbiology 165 (2014) 852e856
capture and rearrangement of resistance regions. This has been recently demonstrated by the appearance of NDM-1 in ST235 strains [7]. Here we survey P. aeruginosa strains from patients in five hospitals in three cities from Colombia in the period 2005e2008. ST235 appeared suddenly in one of the hospital's ICU about half way through the sample period. Analysis of class 1 integron content revealed several atypical structures and genes. This included the presence of class 1 integrons that are presumptive functional novel transposons. ST235 strains are potent vectors for the rapid spread and evolution of complex resistance loci. 2. Materials and methods 2.1. Strain origins, isolation and characterization P. aeruginosa isolates were collected from five hospitals located in Monteria (San Jer onimo Hospital, Clínica Zayma and Clínica Central de Monteria), Cartagena (Bocagrande Hospital) and Barranquilla (Clínica La Asunci on) from 2005 to 2008 following the nosocomial criteria of the Center for Disease Control and Prevention (CDC) in Atlanta for bacteria causing infections. These cities are on the Caribbean coast of Colombia, South America. A total of 179 isolates were recovered. Sixty of 65 isolates were b-lactam resistant to at least one of the drugs tested (see Table 1) and these were analyzed in detail. Five isolates were not recoverable from frozen stocks. Clonal relatedness was evaluated through random amplification of polymorphic DNA (RAPD) analysis, PFGE as a confirmatory method [8] and MLST [9]. Susceptibility profiles were determined by agar diffusion [10]. 2.2. DNA sequencing and genetic characterization Isolates were screened for the intI1 and resistance genes in class 1 integrons using methods and primers previously described [11,12]. The presence of IS26 was assessed as outlined previously [13]. Screening for plasmid DNA was done by multiplex PCR [14]. A fosmid library for the isolate PS38 was made according to the manufacturer's instructions (Epicentre). Sequence of a fosmid clone harboring the intI1þ gene is in Genbank (accession number KF040452). 3. Results 3.1. Isolate isolation and class 1 integron screening Sixty multi drug resistant P. aeruginosa isolates were recovered from individual patients admitted to the five surveyed hospitals. All 60 isolates were tested for the presence of the intI1 gene associated with class 1 integrons. Consistent with other studies [13], a high proportion of strains, 30 (50%), were intI1 positive. Of the five hospitals, one, San Jer onimo Hospital in Monteria (population ~ 400,000), had the single biggest number of isolates (29) and 15 of the intI1þ isolates.
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3.2. Identification of strain types All 15 San Jeronimo intI1þ isolates were examined further for clonal relationships by RAPD and PFGE analysis. On these bases, eight distinct clonal types were identified. One was represented five times and all five were derived from the hospital ICU in the last 18 months of the survey period (Table 1). MLST analysis of these five strains identified them as belonging to P. aeruginosa Sequence Type 235. None of the 15 strains possessed plasmids as determined by Inc-REP PCR analysis (data not shown). All 15 strains were analyzed for the number, location, structure and the type of cassette arrays of the integrons (Table 1). Seven strains, including all five ST235 isolates possessed the array aadA6-gcuD and this was located in Tn6162 in Genomic island 1 (GI1) in the P. aeruginosa chromosome as previously described [8]. 3.3. Characterization of atypical integrons Five strains failed to generate amplicons with primers that target the cassette array region in integrons that possess a 30 CS [15] despite being intI1-positive. This implies the presence of class 1 integrons with atypical structures, in this case lacking that part the tni module normally present when the 30 CS is present. Class 1 integrons with a 30 -CS are considered to be defective transposons since the acquistion of the 30 -CS led to the loss of most of the transposition module common in class 1 integrons in clinical isolates [16]. However, recently, class 1 integrons lacking a 30 -CS but possessing a functional Tn402-like tni module have been identified [12,17]. This was tested for in these strains by the use of PCR primers that targets the Tn402-like tniC gene. Ten strains were negative for tniC including all five strains that failed to generate a PCR product with a 30 -CS primer (Table 1). In contrast, the five ST235 strains were positive for tniC. Using primers that target the array region in class 1 integrons with a complete tni module [12], two types of arrays were recovered. Two isolates had the array blaOxA-101-aacA4-catB3 and the remaining three isolates had a four cassette array comprising blaOxA-101aacA4-catB3-aadA7 (Table 1). The gene cassette aacA4, recovered in the five isolates mentioned above and three other isolates, is likely contributing to the gentamicin resistance phenotype as this association has been previously stablished. In general there is not always a direct correlation between the presence of gene cassettes and particular resistance phenotypes since other mechanisms acting in synergy, such as efflux pumps, can influence overall resistance profiles [13]. However, the presence of the gene OXA-101 does not seems to be absolutely linked to carbapenem resistance in this collection as two isolates are susceptible to them (PS43, PS47). Also they are not associated with 3rd-generation cephalosporin resistance as the all isolates were susceptible to b-lactams inhibitors. A fosmid library was constructed for PS38. Sequencing of a tniC positive clone revealed an integron as shown in Fig. 1. The tni module was complete but comprised a hybrid structure compared to the reference transposon Tn402. Sequence
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E. Martinez et al. / Research in Microbiology 165 (2014) 852e856
Table 1 Multi drug resistant patient isolates of P. aeruginosa from San Jeronimo Hospital between 2005 and 2008. Date Isolate source (month/year)
Isolate RAPD/PFGE/STa tniC IS26 Cassette array tniC
12/05
Cassette array 30 -CS
ATB resistance profilec
PS68
Col-A
tniC integron not present
01/06
ICU-swab wound Swab wound
PS62
Col-B
tniC integron not present
02/06
Ear secretion
PS63
Col-C
tniC integron not present
02/06
Secretion soft tissues ICU-urine
PS64
Col-D
tniC integron not present
aadA6-gcuD IMP, MEM, CAZ, aadB-aacA7-blaOXA2-gcuD GEN, CIP aadA6-gcuD IMP, MEM, AZT, GEN, CIP aadB-aacA4 IMP, MEM, GEN, CIP, FEP No product amplifiedb CAZ
PS71
Col-C
tniC integron not present
aadB-aacA4
PS76
Col-E
tniC integron not present
No product amplifiedb
IMP, MEM, CAZ, GEN, CIP, FEP IMP, MEM, GEN
PS72
Col-D
tniC integron not present
No product amplifiedb
IMP, MEM, CIP
PS74
Col-F
tniC integron not present
aadB-aacA4
GEN, CIP
PS75
Col-E
tniC integron not present
No product amplifiedb
PS38
Col-H/ST235
þ
blaOxA-101-aacA4-catB3
aadA6-gcuD dfrA17-aadA5
PS43
Col-H/ST235
þ
þ
blaOxA-101-aacA4-catB3-aadA7 aadA6-gcuD
IMP, MEM, CAZ, GEN, CIP, FEP IMP, AZT, GEN, CIP, FEP GEN, CIP. FEP
PS47
Col-H/ST235
þ
þ
blaOxA-101-aacA4-catB3
aadA6-gcuD
AZT, GEN, CIP, FEP
PS45
Col-G
þ
tniC integron not present
No product amplifiedb
PS50
Col-H/ST235
þ
blaOxA-101-aacA4-catB3-aadA7 aadA6-gcuD
IMP, MEM, CAZ, GEN, CIP, FEP IMP, GEN, CIP. FEP
PS53
Col-H/ST235
þ
þ
blaOxA-101-aacA4-catB3-aadA7 aadA6-gcuD
03/06 03/06 03/06 04/06 05/06 05/07 08/07 09/07 09/07 10/07 11/08
ICU-bronchial secretion Secretion soft tissues Secretion soft tissues Bronchial secretion ICU-bronchial secretion ICU-bronchial secretion ICU-urinary catheter Tip of catheter ICU-bronchial secretion ICU-bronchial secretion
IMP, MEM, AZT, GEN, CIP, FEP
Strains were designated as intI1 positive on the basis of generation of a PCR amplicon of the size predicted for the primers HS915 and HS916 that target the intI1 gene [11]. a All strains were analyzed by RAPD and PFGE analysis. Strains with a “Col” designation were identified as different/identical clones where the following letter is different/the same. Strains identified as ST235 were 96% similar by PFGE profiles and were distinct from the other strain profiles. MLST analysis of these strains confirmed status as Sequence Type (ST) 235. Only isolates from ST235 were epidemiologically related according to data recovered. b PCR with primers targeting, respectively, the 50 -CS and 30 -CS, failed to generate an amplicon. The location and structure of these integrons is therefore not known. c ATB, Antibiotic; IMP, Imipenem; MEM, Meropenem; CAZ, Ceftazidime:AZT, Aztreonam; GEN, Gentamacin; CIP, Ciprofloxacin; FEP, Cefepime. Other antibiotic tested: Piperacillin, Piperacillin-Tazobactam, Ticarcillin, Ticarcillin-Clavulanic acid, Amikacin, Tobramicin, Levofloxacin, Norfloxacin. Only Resistant isolates defined by CLSI [10] were kept.
comparisons would suggest that the hybrid arose via a sitespecific resolution event as previously described [17]. The region from the transposon resolution site to IRt (Fig. 1) was a best match (87% identity across the tni module) to Tn402. This implies that recombination has occurred with a yet to be
identified transposon. Sequence analysis beyond the transposon the IRi inverted repeat revealed 98% DNA identity to the Pseudomonas stutzeri chromosomal gene PSTAB_2464 suggesting a chromosomal location for this integron/transposon. Based on the information from PS38, PCR analysis
Fig. 1. Structure of class 1 integron linked to the tni module in PS38. The filled vertical rectangles indicate inverted repeats (IRs) as shown. Filled horizontal arrows represent genes and the direction of transcription. Double arrows indicate the extent of regions with the named percentage similarity to Tn402. The sequence of the tniC gene in PS38 was 100% identical to tniC from Tn5090 (identical to Tn402) [20]. However, the sequence from the tniQ gene to IRt presented a level of DNA identity with Tn402 of approximately 87%. Loss of identity has occurred at the res site (R1 and R2) between the tniC and the tniQ genes implying a site-specific resolution event between different tni modules. This has generated a new hybrid tni module compared to Tn402. PSTAB_2464 is a chromosomal gene.
E. Martinez et al. / Research in Microbiology 165 (2014) 852e856
with suitable primers on the remaining four tniC positive clones followed by sequencing, revealed a tni module and location identical to that seen in PS38. 3.4. Presence of IS26 in P. aeruginosa The insertion sequence IS26 has a growing role in the evolution of resistance loci in the Enterobacteriacae [18]. Recently, we observed the appearance of this IS in P. aeruginosa clinical isolates where it appears to influence resistance to some b-lactams by an unusual mechanism [13]. We therefore tested for the presence of IS26 in the 15 intI1-positive strains from San Jer onimo hospital. The IS was present in four strains, three of which were ST235 isolates (Table 1). IS26 only appeared in the strains towards the end of the sample period. 4. Discussion Our data highlights disturbing trends in the evolution of antibiotic resistance as it relates to P. aeruginsosa. Firstly, it is evident that the highly successful Sequence Type ST235 is continuing its global spread. ST235 appeared in the San Jeronimo ICU in 2007 and became established in this location at the expense of the more diverse range of strains that were present in the hospital including in the ICU up to this point. This is consistent with another study in Colombia [5] that found ST235 strains appearing abruptly in 2007. In this case, ST235 strains appeared in hospitals in Cali and Pereira, both being geographically distinct from the Caribbean coast suggesting appearance in Colombia generally around this time. We predict that ST235 has become more prevalent in Colombia since 2007/2008. Many epidemiological studies are carried out in the context of the spread of particular resistance genes. In P. aeruginosa such studies increasingly show a growing propensity for clinically important genes to be found in ST235 isolates [5,6]. The five ST235 isolates recovered here were insightful in demonstrating that the same ST can display different genotypes with respect to complex resistance loci. For example, one of the five isolates, PS38, possessed a third integron not present in the other four. This integron, the location of which is unknown, carried the cassette array dfrA17-aadA5 which is common in integrons from the Enterobacteriacae. Also, there were two forms of the array in the tni-linked integron. One version possessed an aadA7 gene and the second did not (Table 1). The timing of these events is not known but it is possible that these differences represent “real time” changes within the ICU over the sampled time period. Given that class 1 integrons possess specific mechanisms that facilitate rearrangements under stress conditions, including in an antibiotic context [19] such rapid adaptation is clearly possible. Whether the events that occurred here did so in real time or not, it is that virulent and highly dispersed “sequence types” can rearrange clinically important genes by LGT at disturbingly high rates. There are two other aspects of the genetic architecture of the ST235 isolates that emphasizes the trend to increasingly
855
adaptable virulent strains. One of these is the acquisition of multi drug resistance class 1 integrons that are also functional transposons. The more typical class 1 integron in pathogens has a 30 -CS in place of a functional tni module that means that these integrons cannot transpose autonomously. The version seen here however can putatively selftranspose thereby increasing the potential for the spread of resistance in ST235. In addition, Tni proteins can act in trans. Thus the presence of this integron can facilitate the movement of any class 1 integron in the same cell, including the more common tni-defective type [18,20]. Since these strains lack detectable plasmids, it is possible that the gain/ loss of the dfrA17-aadA5 containing integron in PS38 was facilitated by the presence of this functional integron/transposon. The second notable feature of the ST235 strains is the presence of IS26 in three of them. IS26 is capable of mobilizing genes in novel ways [13,18]. It is very common in the Enterobactericaea [18] but rare to date in P. aeruginosa. When present in this species however it appears capable of influencing resistance profiles in ways that are unpredictable, especially when acting cooperatively with other mutations in the cell [13,20]. Movement of this IS into ST235 is another concerning trend in managing the global spread of antibiotic resistance. Funding This work was supported by University of Technology, Sydney, postgraduate scholarship to EM and the Research Division of the El Bosque University, Sinu´ University and Departamento Administrativo de Ciencia, Tecnologia e Innovacion COLCIENCIAS proyect 1283-344-19088. Conflict of interest The authors declare no conflict of interest. References [1] Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! an update from the infectious diseases society of America. Clin Infect Dis 2009;48:1e12. [2] Sardelic S, Bedenic B, Colinon-Dupuich C, Orhanovic S, Bosnjak Z, Plecko V, et al. Infrequent finding of metallo-b-lactamase VIM-2 in carbapenem-resistant Pseudomonas aeruginosa strains from croatia. Antimicrob Agents Chemother 2012;56:2746e9. [3] Liakopoulos A, Mavroidi A, Katsifas E, Theodosiou A, Karagouni A, Miriagou V, et al. Carbapenemase-producing Pseudomonas aeruginosa from central Greece: molecular epidemiology and genetic analysis of class I integrons. BMC Infect Dis 2013;13:505. [4] Cho HH, Kwon KC, Sung JY, Koo SH. Prevalence and genetic analysis of multidrug-resistant Pseudomonas aeruginosa ST235 isolated from a hospital in Korea, 2008e2012. Ann Clin Lab Sci 2013;43:414e9. [5] Cuzon G, Naas T, Villegas M-V, Correa A, Quinn JP, Nordmann P. Wide dissemination of Pseudomonas aeruginosa producing b-lactamase blaKPC-2 gene in Colombia. Antimicrob Agents Chemother 2011;55:5350e3. [6] Edelstein MV, Skleenova EN, Shevchenko OV, D'Souza JW, Tapalski DV, Azizov IS, et al. Spread of extensively resistant VIM-2positive ST235 Pseudomonas aeruginosa in Belarus, Kazakhstan, and
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[7]
[8]
[9]
[10] [11]
[12]
[13]
E. Martinez et al. / Research in Microbiology 165 (2014) 852e856 Russia: a longitudinal epidemiological and clinical study. Lancet Infect Dis 2013;13:867e76. Carattoli A, Fortini D, Galetti R, Garcia-Fernandez A, Nardi G, Orazo D, et al. Isolation of NDM-1-producing Pseudomonas aeruginosa sequence type ST235 from a stem cell transplant patient in Italy, May 2013. Euro Surveill 2013;18. Martinez E, Marquez C, Ingold A, Merlino J, Djordjevic SP, Stokes HW, et al. Diverse mobilized class 1 integrons are common in the chromosome of pathogenic Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 2012;56:2169e72. Curran B, Jonas D, Grundmann H, Pitt T, Dowson CG. Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa. J Clin Microbiol 2004;42:5644e9. CLSI. Performance standards for antimicrobial susceptibility testing. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. Marquez C, Labbate M, Raymondo C, Fernandez J, Gestal AM, Holley M, et al. Urinary tract infections in a South American population: dynamic spread of class 1 integrons and multidrug resistance by homologous and site-specific recombination. J Clin Microbiol 2008;46:3417e25. Betteridge T, Partridge SR, Iredell JR, Stokes HW. Genetic context and structural diversity of class 1 integrons from human commensal bacteria in a hospital intensive care unit. Antimicrob Agents Chemother 2011;55:3939e43. Martinez E, Perez JE, Marquez C, Vilacoba E, Centron D, Leal AL, et al. Emerging and existing mechanisms co-operate in generating diverse b-
[14]
[15]
[16] [17]
[18]
[19]
[20]
lactam resistance phenotypes in geographically dispersed and genetically disparate Pseudomonas aeruginosa strains. J Glob Antimicrob Resist 2013;1:135e42. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219e28. Levesque C, Piche L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995;39:185e91. Brown H, Stokes H, Hall R. The integrons In0, In2, and In5 are defective transposon derivatives. J Bacteriol 1996;178:4429e37. Labbate M, Chowdhury PR, Stokes HW. A class 1 integron present in a human commensal has a hybrid transposition module compared to Tn402: evidence of interaction with Mobile DNA from natural environments. J Bacteriol 2008;190:5318e27. Cain AK, Liu X, Djordjevic SP, Hall RM. Transposons related to Tn1696 in IncHI2 plasmids in multiply antibiotic resistant Salmonella enterica serovar typhimurium from Australian animals. Microb Drug Resist 2010;16:197e202. Hocquet D, Llanes C, Thouverez M, Kulasekara HD, Bertrand X, Plesiat P, et al. Evidence for induction of integron-based antibiotic resistance by the SOS response in a clinical setting. Plos Pathog 2012;8:e1002778. Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clin Infect Dis 2002;34:634e40.
Brief note
Establishment and multi drug resistance evolution of ST235 Pseudomonas aeruginosa strains in the intensive care unit of a Colombian hospital Elena Martinez a,*, Javier Escobar Perez b, Francisco Buelvas c, Catalina Tovar c, Natasha Vanegas a,b, H.W. Stokes a a
The ithree Institute, University of Technology, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia b Laboratorio de Genetica Molecular Bacteriana, Universidad El Bosque, Bogota, Colombia c Grupo de Resistencia Bacteriana y Enfermedades Tropicales, Universidad del Sinu´, Monteria-Cordoba, Colombia Received 14 May 2014; accepted 21 October 2014 Available online 1 November 2014
Abstract Drug resistant Pseudomonas aeruginosa represents a therapeutic challenge. To assess the diversity of P. aeruginosa antibiotic resistant variants, isolates were recovered from hospital patients in Colombia. Thirty of 60 isolates contained class 1 integrons and five were of Sequence Type ST235 having appeared in a single intensive care unit. All five possessed an unusual integron but showed differences in gene cassette content and the presence/absence of insertion sequence IS26. This showed that differences can arise rapidly, even within a single ICU. Also, the emergence of IS26 in P. aeruginosa is contributing to the evolution of resistance in this bacterium. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Class 1 integron; Pseudomonas aeruginosa ST235; IS26; Antibiotic resistance; Hospital acquired infections
1. Introduction Pseudomonas aeruginosa is one of the ESKAPE group of pathogens [1] and is a bacterium that is increasingly recalcitrant to antibiotic therapy. P. aeruginosa is also a significant opportunistic pathogen that manifests in a number of infection contexts most notably in relation to burns victims, cystic fibrosis sufferers and intensive care unit (ICU) patients. One reason P. aeruginosa is increasingly difficult to treat with antibiotics is that it possesses regions of great genomic plasticity indicating substantial amounts of lateral gene transfer (LGT). This LGT can be facilitated by several mobilizing elements including plasmids, transposons and integrons. Many * Corresponding author. E-mail addresses: [email protected] (E. Martinez), [email protected] (J.E. Perez), [email protected] (F. Buelvas), [email protected] (C. Tovar), [email protected] (N. Vanegas), [email protected] (H.W. Stokes).
of these elements and linked genes frequently cluster in chromosomally located genomic islands. These strain specific regions can comprise hundreds of kb in pathogenic strains. Notwithstanding the high level of genome variability it is evident that particular sequence types (ST), as defined by multi locus sequence typing (MLST), appear to be particularly infectious and virulent as measured by their global spread in clinical settings. One of the best examples of this is ST235 which has become broadly established across Europe [2,3]. The clone is also reported in other parts of the world [4] including South America [5] although less commonly. ST235 isolates are commonly associated with the dissemination of particular resistance genes. This is especially true of genes conferring resistance to clinically important b-lactam antibiotics. For example VIM producers have become prevalent in parts of Europe as a consequence of the spread of ST235 clones [3,6]. ST235 strains should not be considered identical however since resistance gene content can vary greatly as the plasticity of P. aeruginosa has allowed on going
http://dx.doi.org/10.1016/j.resmic.2014.10.011 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
E. Martinez et al. / Research in Microbiology 165 (2014) 852e856
capture and rearrangement of resistance regions. This has been recently demonstrated by the appearance of NDM-1 in ST235 strains [7]. Here we survey P. aeruginosa strains from patients in five hospitals in three cities from Colombia in the period 2005e2008. ST235 appeared suddenly in one of the hospital's ICU about half way through the sample period. Analysis of class 1 integron content revealed several atypical structures and genes. This included the presence of class 1 integrons that are presumptive functional novel transposons. ST235 strains are potent vectors for the rapid spread and evolution of complex resistance loci. 2. Materials and methods 2.1. Strain origins, isolation and characterization P. aeruginosa isolates were collected from five hospitals located in Monteria (San Jer onimo Hospital, Clínica Zayma and Clínica Central de Monteria), Cartagena (Bocagrande Hospital) and Barranquilla (Clínica La Asunci on) from 2005 to 2008 following the nosocomial criteria of the Center for Disease Control and Prevention (CDC) in Atlanta for bacteria causing infections. These cities are on the Caribbean coast of Colombia, South America. A total of 179 isolates were recovered. Sixty of 65 isolates were b-lactam resistant to at least one of the drugs tested (see Table 1) and these were analyzed in detail. Five isolates were not recoverable from frozen stocks. Clonal relatedness was evaluated through random amplification of polymorphic DNA (RAPD) analysis, PFGE as a confirmatory method [8] and MLST [9]. Susceptibility profiles were determined by agar diffusion [10]. 2.2. DNA sequencing and genetic characterization Isolates were screened for the intI1 and resistance genes in class 1 integrons using methods and primers previously described [11,12]. The presence of IS26 was assessed as outlined previously [13]. Screening for plasmid DNA was done by multiplex PCR [14]. A fosmid library for the isolate PS38 was made according to the manufacturer's instructions (Epicentre). Sequence of a fosmid clone harboring the intI1þ gene is in Genbank (accession number KF040452). 3. Results 3.1. Isolate isolation and class 1 integron screening Sixty multi drug resistant P. aeruginosa isolates were recovered from individual patients admitted to the five surveyed hospitals. All 60 isolates were tested for the presence of the intI1 gene associated with class 1 integrons. Consistent with other studies [13], a high proportion of strains, 30 (50%), were intI1 positive. Of the five hospitals, one, San Jer onimo Hospital in Monteria (population ~ 400,000), had the single biggest number of isolates (29) and 15 of the intI1þ isolates.
853
3.2. Identification of strain types All 15 San Jeronimo intI1þ isolates were examined further for clonal relationships by RAPD and PFGE analysis. On these bases, eight distinct clonal types were identified. One was represented five times and all five were derived from the hospital ICU in the last 18 months of the survey period (Table 1). MLST analysis of these five strains identified them as belonging to P. aeruginosa Sequence Type 235. None of the 15 strains possessed plasmids as determined by Inc-REP PCR analysis (data not shown). All 15 strains were analyzed for the number, location, structure and the type of cassette arrays of the integrons (Table 1). Seven strains, including all five ST235 isolates possessed the array aadA6-gcuD and this was located in Tn6162 in Genomic island 1 (GI1) in the P. aeruginosa chromosome as previously described [8]. 3.3. Characterization of atypical integrons Five strains failed to generate amplicons with primers that target the cassette array region in integrons that possess a 30 CS [15] despite being intI1-positive. This implies the presence of class 1 integrons with atypical structures, in this case lacking that part the tni module normally present when the 30 CS is present. Class 1 integrons with a 30 -CS are considered to be defective transposons since the acquistion of the 30 -CS led to the loss of most of the transposition module common in class 1 integrons in clinical isolates [16]. However, recently, class 1 integrons lacking a 30 -CS but possessing a functional Tn402-like tni module have been identified [12,17]. This was tested for in these strains by the use of PCR primers that targets the Tn402-like tniC gene. Ten strains were negative for tniC including all five strains that failed to generate a PCR product with a 30 -CS primer (Table 1). In contrast, the five ST235 strains were positive for tniC. Using primers that target the array region in class 1 integrons with a complete tni module [12], two types of arrays were recovered. Two isolates had the array blaOxA-101-aacA4-catB3 and the remaining three isolates had a four cassette array comprising blaOxA-101aacA4-catB3-aadA7 (Table 1). The gene cassette aacA4, recovered in the five isolates mentioned above and three other isolates, is likely contributing to the gentamicin resistance phenotype as this association has been previously stablished. In general there is not always a direct correlation between the presence of gene cassettes and particular resistance phenotypes since other mechanisms acting in synergy, such as efflux pumps, can influence overall resistance profiles [13]. However, the presence of the gene OXA-101 does not seems to be absolutely linked to carbapenem resistance in this collection as two isolates are susceptible to them (PS43, PS47). Also they are not associated with 3rd-generation cephalosporin resistance as the all isolates were susceptible to b-lactams inhibitors. A fosmid library was constructed for PS38. Sequencing of a tniC positive clone revealed an integron as shown in Fig. 1. The tni module was complete but comprised a hybrid structure compared to the reference transposon Tn402. Sequence
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E. Martinez et al. / Research in Microbiology 165 (2014) 852e856
Table 1 Multi drug resistant patient isolates of P. aeruginosa from San Jeronimo Hospital between 2005 and 2008. Date Isolate source (month/year)
Isolate RAPD/PFGE/STa tniC IS26 Cassette array tniC
12/05
Cassette array 30 -CS
ATB resistance profilec
PS68
Col-A
tniC integron not present
01/06
ICU-swab wound Swab wound
PS62
Col-B
tniC integron not present
02/06
Ear secretion
PS63
Col-C
tniC integron not present
02/06
Secretion soft tissues ICU-urine
PS64
Col-D
tniC integron not present
aadA6-gcuD IMP, MEM, CAZ, aadB-aacA7-blaOXA2-gcuD GEN, CIP aadA6-gcuD IMP, MEM, AZT, GEN, CIP aadB-aacA4 IMP, MEM, GEN, CIP, FEP No product amplifiedb CAZ
PS71
Col-C
tniC integron not present
aadB-aacA4
PS76
Col-E
tniC integron not present
No product amplifiedb
IMP, MEM, CAZ, GEN, CIP, FEP IMP, MEM, GEN
PS72
Col-D
tniC integron not present
No product amplifiedb
IMP, MEM, CIP
PS74
Col-F
tniC integron not present
aadB-aacA4
GEN, CIP
PS75
Col-E
tniC integron not present
No product amplifiedb
PS38
Col-H/ST235
þ
blaOxA-101-aacA4-catB3
aadA6-gcuD dfrA17-aadA5
PS43
Col-H/ST235
þ
þ
blaOxA-101-aacA4-catB3-aadA7 aadA6-gcuD
IMP, MEM, CAZ, GEN, CIP, FEP IMP, AZT, GEN, CIP, FEP GEN, CIP. FEP
PS47
Col-H/ST235
þ
þ
blaOxA-101-aacA4-catB3
aadA6-gcuD
AZT, GEN, CIP, FEP
PS45
Col-G
þ
tniC integron not present
No product amplifiedb
PS50
Col-H/ST235
þ
blaOxA-101-aacA4-catB3-aadA7 aadA6-gcuD
IMP, MEM, CAZ, GEN, CIP, FEP IMP, GEN, CIP. FEP
PS53
Col-H/ST235
þ
þ
blaOxA-101-aacA4-catB3-aadA7 aadA6-gcuD
03/06 03/06 03/06 04/06 05/06 05/07 08/07 09/07 09/07 10/07 11/08
ICU-bronchial secretion Secretion soft tissues Secretion soft tissues Bronchial secretion ICU-bronchial secretion ICU-bronchial secretion ICU-urinary catheter Tip of catheter ICU-bronchial secretion ICU-bronchial secretion
IMP, MEM, AZT, GEN, CIP, FEP
Strains were designated as intI1 positive on the basis of generation of a PCR amplicon of the size predicted for the primers HS915 and HS916 that target the intI1 gene [11]. a All strains were analyzed by RAPD and PFGE analysis. Strains with a “Col” designation were identified as different/identical clones where the following letter is different/the same. Strains identified as ST235 were 96% similar by PFGE profiles and were distinct from the other strain profiles. MLST analysis of these strains confirmed status as Sequence Type (ST) 235. Only isolates from ST235 were epidemiologically related according to data recovered. b PCR with primers targeting, respectively, the 50 -CS and 30 -CS, failed to generate an amplicon. The location and structure of these integrons is therefore not known. c ATB, Antibiotic; IMP, Imipenem; MEM, Meropenem; CAZ, Ceftazidime:AZT, Aztreonam; GEN, Gentamacin; CIP, Ciprofloxacin; FEP, Cefepime. Other antibiotic tested: Piperacillin, Piperacillin-Tazobactam, Ticarcillin, Ticarcillin-Clavulanic acid, Amikacin, Tobramicin, Levofloxacin, Norfloxacin. Only Resistant isolates defined by CLSI [10] were kept.
comparisons would suggest that the hybrid arose via a sitespecific resolution event as previously described [17]. The region from the transposon resolution site to IRt (Fig. 1) was a best match (87% identity across the tni module) to Tn402. This implies that recombination has occurred with a yet to be
identified transposon. Sequence analysis beyond the transposon the IRi inverted repeat revealed 98% DNA identity to the Pseudomonas stutzeri chromosomal gene PSTAB_2464 suggesting a chromosomal location for this integron/transposon. Based on the information from PS38, PCR analysis
Fig. 1. Structure of class 1 integron linked to the tni module in PS38. The filled vertical rectangles indicate inverted repeats (IRs) as shown. Filled horizontal arrows represent genes and the direction of transcription. Double arrows indicate the extent of regions with the named percentage similarity to Tn402. The sequence of the tniC gene in PS38 was 100% identical to tniC from Tn5090 (identical to Tn402) [20]. However, the sequence from the tniQ gene to IRt presented a level of DNA identity with Tn402 of approximately 87%. Loss of identity has occurred at the res site (R1 and R2) between the tniC and the tniQ genes implying a site-specific resolution event between different tni modules. This has generated a new hybrid tni module compared to Tn402. PSTAB_2464 is a chromosomal gene.
E. Martinez et al. / Research in Microbiology 165 (2014) 852e856
with suitable primers on the remaining four tniC positive clones followed by sequencing, revealed a tni module and location identical to that seen in PS38. 3.4. Presence of IS26 in P. aeruginosa The insertion sequence IS26 has a growing role in the evolution of resistance loci in the Enterobacteriacae [18]. Recently, we observed the appearance of this IS in P. aeruginosa clinical isolates where it appears to influence resistance to some b-lactams by an unusual mechanism [13]. We therefore tested for the presence of IS26 in the 15 intI1-positive strains from San Jer onimo hospital. The IS was present in four strains, three of which were ST235 isolates (Table 1). IS26 only appeared in the strains towards the end of the sample period. 4. Discussion Our data highlights disturbing trends in the evolution of antibiotic resistance as it relates to P. aeruginsosa. Firstly, it is evident that the highly successful Sequence Type ST235 is continuing its global spread. ST235 appeared in the San Jeronimo ICU in 2007 and became established in this location at the expense of the more diverse range of strains that were present in the hospital including in the ICU up to this point. This is consistent with another study in Colombia [5] that found ST235 strains appearing abruptly in 2007. In this case, ST235 strains appeared in hospitals in Cali and Pereira, both being geographically distinct from the Caribbean coast suggesting appearance in Colombia generally around this time. We predict that ST235 has become more prevalent in Colombia since 2007/2008. Many epidemiological studies are carried out in the context of the spread of particular resistance genes. In P. aeruginosa such studies increasingly show a growing propensity for clinically important genes to be found in ST235 isolates [5,6]. The five ST235 isolates recovered here were insightful in demonstrating that the same ST can display different genotypes with respect to complex resistance loci. For example, one of the five isolates, PS38, possessed a third integron not present in the other four. This integron, the location of which is unknown, carried the cassette array dfrA17-aadA5 which is common in integrons from the Enterobacteriacae. Also, there were two forms of the array in the tni-linked integron. One version possessed an aadA7 gene and the second did not (Table 1). The timing of these events is not known but it is possible that these differences represent “real time” changes within the ICU over the sampled time period. Given that class 1 integrons possess specific mechanisms that facilitate rearrangements under stress conditions, including in an antibiotic context [19] such rapid adaptation is clearly possible. Whether the events that occurred here did so in real time or not, it is that virulent and highly dispersed “sequence types” can rearrange clinically important genes by LGT at disturbingly high rates. There are two other aspects of the genetic architecture of the ST235 isolates that emphasizes the trend to increasingly
855
adaptable virulent strains. One of these is the acquisition of multi drug resistance class 1 integrons that are also functional transposons. The more typical class 1 integron in pathogens has a 30 -CS in place of a functional tni module that means that these integrons cannot transpose autonomously. The version seen here however can putatively selftranspose thereby increasing the potential for the spread of resistance in ST235. In addition, Tni proteins can act in trans. Thus the presence of this integron can facilitate the movement of any class 1 integron in the same cell, including the more common tni-defective type [18,20]. Since these strains lack detectable plasmids, it is possible that the gain/ loss of the dfrA17-aadA5 containing integron in PS38 was facilitated by the presence of this functional integron/transposon. The second notable feature of the ST235 strains is the presence of IS26 in three of them. IS26 is capable of mobilizing genes in novel ways [13,18]. It is very common in the Enterobactericaea [18] but rare to date in P. aeruginosa. When present in this species however it appears capable of influencing resistance profiles in ways that are unpredictable, especially when acting cooperatively with other mutations in the cell [13,20]. Movement of this IS into ST235 is another concerning trend in managing the global spread of antibiotic resistance. Funding This work was supported by University of Technology, Sydney, postgraduate scholarship to EM and the Research Division of the El Bosque University, Sinu´ University and Departamento Administrativo de Ciencia, Tecnologia e Innovacion COLCIENCIAS proyect 1283-344-19088. Conflict of interest The authors declare no conflict of interest. References [1] Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! an update from the infectious diseases society of America. Clin Infect Dis 2009;48:1e12. [2] Sardelic S, Bedenic B, Colinon-Dupuich C, Orhanovic S, Bosnjak Z, Plecko V, et al. Infrequent finding of metallo-b-lactamase VIM-2 in carbapenem-resistant Pseudomonas aeruginosa strains from croatia. Antimicrob Agents Chemother 2012;56:2746e9. [3] Liakopoulos A, Mavroidi A, Katsifas E, Theodosiou A, Karagouni A, Miriagou V, et al. Carbapenemase-producing Pseudomonas aeruginosa from central Greece: molecular epidemiology and genetic analysis of class I integrons. BMC Infect Dis 2013;13:505. [4] Cho HH, Kwon KC, Sung JY, Koo SH. Prevalence and genetic analysis of multidrug-resistant Pseudomonas aeruginosa ST235 isolated from a hospital in Korea, 2008e2012. Ann Clin Lab Sci 2013;43:414e9. [5] Cuzon G, Naas T, Villegas M-V, Correa A, Quinn JP, Nordmann P. Wide dissemination of Pseudomonas aeruginosa producing b-lactamase blaKPC-2 gene in Colombia. Antimicrob Agents Chemother 2011;55:5350e3. [6] Edelstein MV, Skleenova EN, Shevchenko OV, D'Souza JW, Tapalski DV, Azizov IS, et al. Spread of extensively resistant VIM-2positive ST235 Pseudomonas aeruginosa in Belarus, Kazakhstan, and
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