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Multilocus Sequence Types of Carbapenem-Resistant Pseudomonas aeruginosa in Singapore Carrying Metallo Lactamase Genes, Including the Novel blaIMP-26 Gene

American Society for Microbiology
Journal of Clinical Microbiology
Authors:
Tiptiya Koh at Sukhothai Thammathirat Open University
Tiptiya Koh
Cheng Teng Khoo
Cheng Teng Khoo
Cheng Teng Khoo
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Thean Yen Tan at Changi General Hospital
Thean Yen Tan
Mohamed Amir Bin Mohamed Arshad
Mohamed Amir Bin Mohamed Arshad
Mohamed Amir Bin Mohamed Arshad
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Abstract and Figures

Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-beta-lactamase genes, including bla(IMP-1), bla(IMP-7), bla(VIM-2), bla(VIM-6), and the novel bla(IMP-26). Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.
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JOURNAL OF CLINICAL MICROBIOLOGY, July 2010, p. 2563–2564 Vol. 48, No. 7
0095-1137/10/$12.00 doi:10.1128/JCM.01905-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Multilocus Sequence Types of Carbapenem-Resistant
Pseudomonas aeruginosa in Singapore Carrying
Metallo-␤-Lactamase Genes, Including
the Novel bla
IMP-26
Gene
䌤
Tse Hsien Koh,*
1
Cheng Teng Khoo,
1
Thuan Tong Tan,
2
Mohamed Amir Bin Mohamed Arshad,
3
Li Ping Ang,
3
Lee Jin Lau,
3
Li-Yang Hsu,
4
and Eng Eong Ooi
5
Department of Pathology, Singapore General Hospital, Outram Road, 169608 Singapore
1
; Department of Infectious Diseases,
Singapore General Hospital, Outram Road, 169608 Singapore
2
; Temasek Applied Science School, Temasek Polytechnic,
21 Tamplines Avenue 1, 529757 Singapore
3
; Department of Medicine, Yong Loo Lin School of Medicine, National University of
Singapore, 5 Lower Kent Ridge Road, 119074 Singapore
4
; and Duke-NUS Graduate Medical School,
8 College Road, 169857 Singapore
5
Received 26 September 2009/Returned for modification 13 October 2009/Accepted 26 April 2010
Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-␤-
lactamase genes, including bla
IMP-1
,bla
IMP-7
,bla
VIM-2
,bla
VIM-6
, and the novel bla
IMP-26
. Multilocus sequence
typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the
epidemic clones from 2000 have not become established.
Carbapenem-resistant Pseudomonas aeruginosa is an in-
creasing problem worldwide. While many underlying mecha-
nisms may account for carbapenem resistance in this species,
the possession of metallo-␤-lactamase (MBL) genes is of par-
ticular concern because these enzymes are able to hydrolyze all
␤-lactam antimicrobials with the exception of aztreonam. In
addition, these genes may be mobilized and transferred be-
tween different species of bacteria. We conducted a study in
2008 to investigate if there were any changes in the epidemi-
ology of P. aeruginosa isolates containing MBL genes in our
hospital compared to results from an earlier survey carried out
in 2000 (3).
Of 2,552 nonduplicate P. aeruginosa organisms isolated in
2008, 123 isolates were imipenem resistant. Of these, 11 were
positive for MBL production by imipenem-EDTA disk diffu-
sion (5). Nine of these yielded a product by multiplex PCR for
MBL genes (2). The individual MBL genes were then ampli-
fied and sequenced. The clonal relationship between isolates
with MBL genes was determined by pulsed-field gel electro-
phoresis (PFGE) of chromosomal DNA restricted with SpeI
(3). The PFGE band patterns were analyzed with Bionumerics
(Applied Maths NV, Sint-Martens-Latem, Belgium), and all
strains with more than 85% similarity were considered to be-
long to the same clone. All strains were further subjected to
multilocus sequence typing (MLST) (1). Because it is a nucleic
acid sequence-based method, MLST is able to characterize
bacterial types in an unambiguous fashion and establish evo-
lutionary relationships between strains better than band-based
methods like PFGE. Representative MBL-producing P. aerugi-
nosa isolates from the 2000 survey were also subjected to
PFGE and MLST. MLST profiles were submitted to eBURST
V3 (http://eburst.mlst.net/) on 10 March 2010. Isolates sharing
six out of seven alleles were assigned to the same BURST
group and can be considered to belong to the same clonal
complex descended from a common founder genotype. The
PFGE, MBL gene sequence, and MLST results are summa-
rized in Fig. 1.
In our previous study, 21 of 2,094 (1.0%) of all nonduplicate
P. aeruginosa isolates in our hospital had MBL genes (3). With
the exception of one isolate with bla
IMP-7
, all other isolates had
bla
IMP-1
and belonged to one of two PFGE clones. Isolates
belonging to clone A had sequences identical to that of the
original bla
IMP-1
first reported in Japan. Four representatives
of clone A isolated from our hospital in 2000 had sequence
type 964 (ST964) by MLST. Isolates belonging to clone B
isolated in 2000 had sequences for variant bla
IMP-1
(bla
IMP-1v
)
with four silent mutations. Three representatives of this clone
from 2000 had ST233 and one had ST742 based on MLST. All
four representatives of clone B belong to the same BURST
group, which was different from that of clone A.
In contrast, in the 2008 survey, 9 of 2,552 (0.35%) nondu-
plicate P. aeruginosa isolates had MBL genes. Unlike the ear-
lier study, there were no large clonal outbreaks. Two isolates
with bla
IMP-1v
had similar PFGE patterns and belonged to the
same BURST group as representative isolates from clone B in
2000.
Two isolates from 2008 with bla
IMP-7
had similar PFGE
patterns and shared the same BURST group. The rest of the
isolates from 2008 had distinct PFGE patterns.
There was a greater diversity of MBL genes compared to the
2000 survey results. In particular, this is the first time that
bla
VIM-2
and bla
VIM-6
have been found in P. aeruginosa in
Singapore. bla
IMP-26
is a novel MBL gene that differs from
bla
IMP-4
at position 145 (G-to-T change). The translated amino
acid sequence differs from IMP-4 at residue 49 (phenylalanine
* Corresponding author. Mailing address: Department of Pathology,
Singapore General Hospital, Outram Road, 169608 Singapore. Phone:
65-63214275. Fax: 65-62226826. E-mail: koh.tse.hsien@sgh.com.sg.
䌤
Published ahead of print on 12 May 2010.
2563
for valine). This sequence has been previously deposited in the
GenBank database as IMP-4 from an Acinetobacter calcoace-
ticus isolate from Malaysia (accession number ABC24668.1).
Three of the isolates in this study (separately containing
bla
VIM-2
,bla
IMP-1
, and bla
IMP-7
) belonged to ST235. This se-
quence type has been described in a VIM-producing P. aerugi-
nosa isolate in Belgrade and is the founder of an international
clonal complex of isolates bearing MBL genes found in several
countries in Europe (6). Recently, an increasing prevalence of
IMP-1-producing P. aeruginosa has been found in Hiroshima,
Japan. This was due entirely to the clonal expansion of only
two lineages, ST235 (BURST group 3) and ST357 (BURST
group 108) (4). This is similar to the situation that existed in
Singapore in 2000, where only two lineages (BURST groups 29
and 44) accounted for the majority of MBL-producing P.
aeruginosa (3).
It is noteworthy that the original fear that a clone of MBL-
producing P. aeruginosa would become established in Singa-
pore has not been realized. The BURST group 29 and 44
lineages from 2000 were represented by only one to two iso-
lates in 2008. The two P. aeruginosa isolates with bla
IMP-7
in
2008 are unrelated to the solitary isolate with bla
IMP-7
from
2000. It has been suggested that P. aeruginosa displays an
epidemic population structure, with a limited number of clones
emerging from a large number of unrelated genotypes (7).
Although we did not correlate our study with hospital infection
control measures, the Japanese data and our own seem to
suggest that controlling the prevalence of MBL-producing P.
aeruginosa may be achieved by preventing the transmission of
specific epidemic clones.
While it is reassuring to note that the prevalence of MBL
producers in carbapenem-resistant P. aeruginosa has not in-
creased, the increased diversity of MBL genes represents a new
cause for concern. We were unable to characterize the gene
responsible for the MBL phenotype in two isolates in this
study, and these may represent novel resistance determinants.
Although clones of MBL-producing P. aeruginosa have not
become established, it seems likely, given the variation of MBL
genes and MLST types in this study, that MBL-producing P.
aeruginosa continues to be introduced to our hospital from
diverse sources.
Nucleotide sequence accession number. The sequence for
bla
IMP-26
was submitted to GenBank under the accession num-
ber GU045307.
We acknowledge Ong Lan Huay for technical assistance.
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FIG. 1. Dendrogram of PFGE patterns of P. aeruginosa isolates with metallo-␤-lactamase genes, showing the year of isolation, MLST sequence
type, and BURST group.
2564 NOTES J. CLIN.MICROBIOL.
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... CRE strains, which exhibit a multidrug-resistant or extensively drug-resistant pattern, are considered a serious threat to public health, and there are few effective treatment options for the resulting infections. We isolated two IMP-4-producing CRECL strains and one IMP-4-producing K. pneumoniae from 259 clinical CRE strains, and it seems that IMP-4 is more prevalent in CRECL strains (9,15); IMP-26 is less reported nationally and mostly found in P. aeruginosa (8,16), but our study found its detection from CRECL (17,18). The results of the conjugation assays showed that only the pIMP4-ECL42 plasmid could be successfully transferred into E. coli 600, probably because IncC is a self-transmis sible plasmid and can mediate the co-transmission of bla IMP-4 and other ARGs (19)(20)(21), and IncC is also one of the most predominant plasmid replicon type carrying the bla IMP-4 (3,22). ...
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Full-text available
  • Feb 2023
Objective To analyze the molecular epidemiology of carbapenem-resistant Enterobacter cloacae complex (CREC) by whole-genome sequencing and to explore its clinical characteristics. Methods Enterobacter cloacae complex isolates collected in a tertiary hospital during 2013–2021 were subjected to whole-genome sequencing to determine the distribution of antimicrobial resistance genes (ARGs), sequence types (STs), and plasmid replicons. A phylogenetic tree of the CREC strains was constructed based on the whole-genome sequences to analyze their relationships. Clinical patient information was collected for risk factor analysis. Results Among the 51 CREC strains collected, bla NDM-1 ( n = 42, 82.4%) was the main carbapenem-hydrolyzing β-lactamase (CHβL), followed by bla IMP-4 ( n = 11, 21.6%). Several other extended-spectrum β-lactamase-encoding genes were also identified, with bla SHV-12 ( n = 30, 58.8%) and bla TEM-1B ( n = 24, 47.1%) being the predominant ones. Multi-locus sequence typing revealed 25 distinct STs, and ST418 ( n = 12, 23.5%) was the predominant clone. Plasmid analysis identified 15 types of plasmid replicons, among which IncHI2 ( n = 33, 64.7%) and IncHI2A ( n = 33, 64.7%) were the main ones. Risk factor analysis showed that intensive care unit (ICU) admission, autoimmune disease, pulmonary infection, and previous corticosteroid use within 1 month were major risk factors for acquiring CREC. Logistic regression analysis showed that ICU admission was an independent risk factor for CREC acquisition and was closely related with acquiring infection by CREC with ST418. Conclusion Bla NDM-1 and bla IMP-4 were the predominant carbapenem resistance genes. ST418 carrying Bla NDM-1 not only was the main clone, but also circulated in the ICU of our hospital during 2019–2021, which highlights the necessity for surveillance of this strain in the ICU. Furthermore, patients with risk factors for CREC acquisition, including ICU admission, autoimmune disease, pulmonary infection, and previous corticosteroid use within 1 month, need to be closely monitored for CREC infection.
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Role of megaplasmids and chromosomal integration in acquisition of CTX-M-encoding genes by Pseudomonas aeruginosa
Article
  • Sep 2022
The genes that encode CTX-M-type ESBLs have diffused extensively among Enterobacterales over the past decades.¹ Despite this epidemic success at the global level, few of these have been detected (blaCTX-M-1,-2,-3,-14,-15and-43) in the distantly related opportunistic pathogen Pseudomonas aeruginosa.1–4 The narrow host range of plasmids carrying the blaCTX-M determinants in Enterobacterales (mainly of incompatibility group IncF) plausibly accounts for the low prevalence of these resistance genes in P. aeruginosa.⁵ On the other hand, little is known about the mobile elements that enable the acquisition of CTX-M enzymes by clinical P. aeruginosa strains. One plasmid close to broad-host-range incompatibility group IncP2 has been found to carry the gene blaCTX-M-2, while another one belonging to IncQ harboured blaCTX-M-3.6,7 The present report describes the structure of a large conjugative plasmid determining a novel CTX-M variant, named CTX-M-206, and the genetic environment of a chromosomally located blaCTX-M-15 gene in a P. aeruginosa isolate present in a second strain.
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Molecular characterization of carbapenem-resistant Pseudomonas aeruginosa clinical isolates in Nepal
Article
  • Jul 2021
Objectives The emergence of carbapenem-resistant Pseudomonas aeruginosa has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of carbapenem-resistant P. aeruginosa strains isolated from hospitals in Nepal. Methods Forty-three carbapenem-resistant P. aeruginosa isolates obtained from patients in two hospitals in Nepal between 2018 and 2020 were analyzed. Their whole genomes were sequenced by next generation sequencing. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Results Of the 43 isolates, 17 harbored genes encoding carbapenemases, including IMP-1, IMP-26, KPC-2, NDM-1, VIM-2 and VIM-5; and 12 harbored genes encoding 16S rRNA methylases, including RmtB4 and RmtF2. The carbapenem-resistant P. aeruginosa isolated in Nepal belonged to various sequence types, including ST235 (5 isolates), ST244 (7 isolates), ST274 (1 isolate), ST357 (10 isolates), ST654 (3 isolates), ST664 (1 isolate), ST773 (1 isolate), ST823 (3 isolates), ST1047 (8 isolates), ST1203 (2 isolates), and ST3453 (2 isolates). Conclusions This is the first molecular epidemiological analysis of carbapenem-resistant P. aeruginosa clinical isolates in Nepal. These findings strongly suggest that P. aeruginosa isolates producing carbapenemases and 16S rRNA methylases have spread throughout medical settings in Nepal.
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Clonal Spread of IMP1-Producing Pseudomonas aeruginosa in Two Hospitals in Singapore
Article
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Thirty-six isolates of carbapenem-resistant Pseudomonas aeruginosa were studied. Pulsed-field gel electrophoresis revealed the presence of two clones. One clone carried a bla(IMP-1) gene identical to that first described in Japan. The other clone carried a bla(IMP-1) variant containing four silent mutations. One isolate with a unique pulsed-field gel electrophoresis pattern contained bla(IMP-7).
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Development of a Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Pseudomonas aeruginosa
Article
Full-text available
A multilocus sequence typing (MLST) scheme has been developed for Pseudomonas aeruginosa which provides molecular typing data that are highly discriminatory and electronically portable between laboratories. MLST data confirm the data from previous studies that suggest that P. aeruginosa is best described as nonclonal but as having an epidemic population. The index of association was 0.17, indicating a freely recombining population; however, there was evidence of clusters of closely related strains or clonal complexes among the members of this population. It is apparent that the sequence types (STs) from single isolates, representing each of the present epidemic clones in the United Kingdom from Liverpool, Manchester, and the West Midlands, are not closely related to each other. This suggests distinct evolutionary origins for each of these epidemic clones in the United Kingdom. Furthermore, these clones are distinct from European clone C. Comparison of the results of MLST with those of toxA typing and serotyping revealed that strains with identical STs may possess different toxA types and diverse serotypes. Given that recombination is important in the population of P. aeruginosa, the lack of a linkage between toxA type and serotype is not surprising and reveals the strength of the MLST approach for obtaining a better understanding of the epidemiology of P. aeruginosa.
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Increased prevalence and clonal dissemination of multidrug-resistant Pseudomonas aeruginosa with the bla(IMP-1) gene cassette in Hiroshima
Article
  • Apr 2009
  • J ANTIMICROB CHEMOTH
The aim of this study was to evaluate the dissemination of metallo-beta-lactamase (MBL)-encoding genes among multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from major hospitals in the Hiroshima region. During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC >or= 16 mg/L). They were screened for resistance to amikacin (MIC >or= 64 mg/L) and ciprofloxacin (MIC >or= 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST). The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla(IMP-1) and bla(VIM-2) genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla(IMP-1) cassettes and a single array in bla(VIM-2) cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla(IMP-1) gene cassette array were most widely distributed in Hiroshima. This study shows a dramatic increase in MBL genes, primarily bla(IMP-1), in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla(IMP-1) gene cassette array appears to be clonally expanding.
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Characterisation of the first VIM metallo - Lactamase-producing Pseudomonas aeruginosa clinical isolate in Serbia
Article
  • Jan 2009
From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa strains were published from Croatia, Poland and Hungary. The aim of this study was to assess the contribution of MBL-production to carbapenem-resistance among P. aeruginosa clinical isolates in the Military Medical Academy (MMA) in Belgrade, Serbia between August 2004 and September 2007. Only one P. aeruginosa isolate with strain number 722 proved MBL-positive that harboured a novel class 1 integron with a bla(VIM-2)-like cassette in the first position, followed by orfD, a putative gene with unknown function. Our data indicate that MBL-producing strains occur at a prevalence of less than 1% among imipenem-nonsusceptible P. aeruginosa clinical isolates in this Belgrade hospital. The newly identified VIM MBL-producing P. aeruginosa strain 722 could be assigned to serotype O11, and it was panresistant to all antimicrobials tested. The isolate displayed sequence type ST235 by multilocus sequence typing which is the founder sequence type of the previously identified international clonal complex CC11 that already contains bla(VIM)-positive isolates from Italy, Greece, Sweden, Hungary and Poland. In conclusion, this is the first report of VIM MBL-producing P. aeruginosa from Serbia and also of the occurrence of such isolates belonging to the international clonal complex CC11 in this country.
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Pseudomonas aeruginosa displays an epidemic population structure
Article
  • Jan 2003
Bacteria can have population structures ranging from the fully sexual to the highly clonal. Despite numerous studies, the population structure of Pseudomonas aeruginosa is still somewhat contentious. We used a polyphasic approach in order to shed new light on this issue. A data set consisting of three outer membrane (lipo)protein gene sequences (oprI, oprL and oprD), a DNA-based fingerprint (amplified fragment length polymorphism), serotype and pyoverdine type of 73 P. aeruginosa clinical and environmental isolates, collected across the world, was analysed using biological data analysis software. We observed a clear mosaicism in the results, non-congruence between results of different typing methods and a microscale mosaic structure in the oprD gene. Hence, in this network, we also observed some clonal complexes characterized by an almost identical data set. The most recent clones exhibited serotypes O1, 6, 11 and 12. No obvious correlation was observed between these dominant clones and habitat or, with the exception of some recent clones, geographical origin. Our results are consistent with, and even clarify, some seemingly contradictory results in earlier epidemiological studies. Therefore, we suggest an epidemic population structure for P. aeruginosa, comparable with that of Neisseria meningitidis, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise.
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Pseudomonas aeruginosa displays an epidemic population structure FIG. 1. Dendrogram of PFGE patterns of P. aeruginosa isolates with metallo--lactamase genes, showing the year of isolation, MLST sequence type, and BURST group
  • 898-911
  • J P Pirnay
  • D De
  • C Vos
  • F Cochez
  • A Bilocq
  • M Vanderkelen
  • B Zizi
  • P Ghysels
  • Cornelis
Pirnay, J. P., D. De Vos, C. Cochez, F. Bilocq, A. Vanderkelen, M. Zizi, B. Ghysels, and P. Cornelis. 2002. Pseudomonas aeruginosa displays an epidemic population structure. Environ. Microbiol. 4:898–911. FIG. 1. Dendrogram of PFGE patterns of P. aeruginosa isolates with metallo--lactamase genes, showing the year of isolation, MLST sequence type, and BURST group. 2564 NOTES J. CLIN. MICROBIOL.