First Survey of Metallo-?-Lactamase Producers in Clinical Isolates of Pseudomonas aeruginosa From a Referral Burn Center in Kurdistan Province.

0.81 MB·English

by  Kalantar, Enayatollah

7
Jundishapur J Nat Pharm Prod.2012; (1):23-26.
Jundishapur Journal of Natural
Pharmaceutical Products
www.jjnpp.com
β
First Survey of Metallo- –Lactamase Producers in Clinical Isolates of
Pseudomonas aeruginosa
From a Referral Burn Center in Kurdistan
Province
Enayatollah Kalantar 1*, Vahideh Torabi 2, Heiman Salimizand 3, Fariborz Soheili 4, Soheila
Beiranvand 5, Mohammad Mehdi Soltan Dallal 6
1 Envirronmental Health Resaerch Center, Kurdistan University of Medical Sciences, Sanandaj, IR Iran
2 Department of Biological Sciences, Islamic Azad University, Sciences Research Branch, Tehran, IR Iran
3 Department of Microbiology, School of Medicine, Kurdistan University of Medical Sciences, Sanandaj, IR Iran
4 School of Medicine, Kurdistan University of Medical Sciences, Sanandaj, IR Iran
5 Qods Hospital, Kurdistan University of Medical Sciences, Sanandaj, IR Iran
6 Division of Microbiology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran
A R T I C L E I N F O A B S T R A C T
Article type: Background:
Treatment of infectious diseases is becoming more challenging with each
Pseudomonas aeruginosa
Original Aricle passing year. This is especially true for infections caused by , an
opportunistic pathogen with the ability to rapidly develop resistance to multiple classes
Article history:
of antibiotics.
Objective: β
Received: 2 Nov 2011 This study was conducted to determine the prevalence of metallo- -lactamase
P. aeruginosa
Revised: 25 Nov 2011 (MBL)–producing strains among multidrug-resistant strains isolated from
Accepted: 5 Dec 2011 burn patients.
Materials and Methods:
The isolates were identified, tested for susceptibility to various
Keywords: β
antimicrobial agents, and screened for the presence of M Ls by using the double-disk
Burn Patients synergy test. The minimal inhibitory concentration of imipenem was determined by
P. aeruginosa
microplate broth dilution method on Mueller-Hinton agar. To detect VIM, SIM, and GIM
β
Metallo- -lactamase MBLs, the isolates were subjected to polymerase chain reaction.
Results: P. aeruginosa
Imipenem Resistance In this study, we identified 100 isolates from 176 clinical specimens
obtained from burn patients. The isolates showed maximum resistance to ampicillin
(100%), ceftazidime (94%), and ceftriaxone (89%). The CLSI-MBL phenotypic test showed
P. aeruginosa
that of the 100 isolates, 22 (22%) were positive for MBL production in the
P. aeruginosa
double-disk synergy test. Of the 22 MBL-positive isolates, 8 were resistant to
imipenem. PCR analysis showed that 8 isolates were positive for blaVIM1. The other genes
blaSIM1 and blaGIM1 were not detected.
Conclusion:
The study results demonstrate the serious therapeutic threat of the spread
P. aeruginosa β
of MBL producers among populations. Metallo- -lactamases were detected
P. aeruginosa
in 22% of imipenem-resistant isolates. Early detection and infection-control
practices are the best antimicrobial strategies for this organism; therefore, systematic
surveillance to detect MBL producers is necessary.
Copyright c2012 DocS.
Implication for health policy/practice/research/medical education:
Pseudomonas aeruginosa
, has showed up as a significant nosocomial pathogen particularly among burn patients. We have very
Pseudomonas aeruginosa
scant information about the current state of infections and their resistance pattern particularly ESBL.
Moreover, with the increased use of prophylactic antibiotics to burn patients, careful surveillance of the changing trend of bacte-
rial organisms and their antibiotic resistance pattern among neonates is warranted.
Corresponding author:
* Dr. Enayatollah Kalantar, Envirronmental Health
Resaerch Center, Kurdistan University of Medical Sciences, Sanandaj, IR
E-mail:
Iran. Tel.: [phone], Fax: [phone], ekalantar@hot-
mail.com
Copyright c2012 DocS.
24 Kalantar E et al. Metallo-β–Lactamase Producers Among Pseudomonas Aeruginosa Isolates
Please cite this paper as:
Kalantari E, Torabi V, Salimizand H, Soheili F, Beiranvand S, Soltan Dallal MM, First Survey of Metallo-β–Lactamase Producers in
Jundishapur J Nat Pharm Prod
Clinical Isolates of Pseudomonas aeruginosa From a Referral Burn Center in Kurdistan Province. .
7
2012; (1):23-6.
1. Background 3-1. Antimicrobial Susceptibility Testing
Pseudomonas aeruginosa
, a versatile pathogen associ- Antimicrobial susceptibility testing was done by the
ated with a broad spectrum of infections in humans and disk-diffusion method on Muller-Hinton agar (Merck,
widely known as an opportunistic organism, is frequent- Germany) (11). The following antibiotics were used: ami-
ly involved in infections among susceptible populations, kacin, gentamicin, carbencillin, ciprofloxacin, ofloxacin,
especially patients with burns (1). Furthermore, this or- cefepime, ceftazidime, cefotaxime, ampicillin, ceftriax-
ganism possesses several virulence factors and is intrin- one, imipenem, and meropenem.
sically resistant to most antimicrobials, a feature that
3-2. Detection of MBL
is also responsible for the difficulty in treating infected
P aeruginosa
patients (2). In the past decade, . strains P aeruginosa
Detection of MBL-producing . strains was
β
showing resistance to multiple -lactam antibiotics, in
β P. ae- performed according to Clinical and Laboratory Stan-
particular, metallo- -lactamase (MBL)–producing P. aeruginosa
ruginosa dards Institute (CLSI) guidelines (11). ATCC
, have become an increasing public health prob-
27853 was used as the negative control. For the double-
lem worldwide (3-5). Furthermore, infection with MBL-
disk synergy test, the inoculum was prepared after
producing organisms is associated with higher rates of
emulsifying 5–6 colonies of the suspected isolate in
mortality, morbidity, and health care costs (6-8).
P. aeruginosa Mueller–Hinton broth, and the turbidity was adjusted to
In , resistance to extended-spectrum
0.5 McFarland opacity standard. A lawn culture was ob-
β
-lactams is mediated by lack of drug penetration, which
tained on Mueller–Hinton agar, and double-disk synergy
may occur due to porin mutations, efflux pumps, or
test was performed.
β
hydrolysis by -lactamases (9). On the basis of molecu-
lar studies, we can classify carbapenem-hydrolyzing 3-3. Determination of Minimal Inhibitory Concentra-
enzymes into 4 groups: A, B, C, and D. The MBLs, which tion
belong to group B, are enzymes that require divalent
cations as cofactors for optimal enzyme activity and are Imipenem minimal inhibitory concentration (MIC)
inhibited by the action of a metal ion chelator. Hospital was determined by microplate (NUNC, Denmark) broth
P. aeruginosa
infections caused by are often difficult to dilution method on Mueller–Hinton agar. Zone diameter
P. ae-
eradicate because the organisms are resistant to drugs. and MIC interpretive standards for breakpoints for
P. aeruginosa ruginosa
Therefore, detection of MBL-producing is published by CLSI were used (11).
crucial to control the spread of resistance and for opti-
3-4 Genotypic Detection of VIM, SIM, and GIM MBL
mal treatment of patients, especially burn patients (10).
There is no information concerning the prevalence of Genes by PCR
P. aeruginosa
MBL-producing strains in Kurdistan prov-
To detect VIM, SIM, and GIM MBL genes, PCR was per-
ince. P. aeruginosa
formed for isolates (12)
2. Objectives
4. Results
We conducted this study to detect MBL-producing
P. aeruginosa
P. aeruginosa In this study, we identified 100 isolates
strains among isolates obtained from burn
among 176 clinical specimens obtained from patients.
patients at Tohid hospital, which is affiliated to Kurdistan
Table 1 P. aeru-
shows the antibiotic-resistance pattern of
University of Medical Sciences and has a burn unit with a
ginosa
isolates. The isolates showed maximum resistance
heavy patient turnover and extensive antibiotic use.
to ampicillin (100%), ceftazidime (94%), and ceftriaxone
3. Materials and Methods (89%). The next step in testing was designed to identify
P. aeruginosa
the MBL-producing strains. In the CLSI-MBL
Between April 2009 and April 2010, we isolated and iden- phenotypic test, of the 100 P. aeruginosa isolates, 22 (22%)
P. aeruginosa
tified 100 strains of in a clinical laboratory were positive for MBL production by the double-disk syn-
at Tohid hospital, Sanandaj, Iran. The strains were identi- ergy test (Figure 1).
P. aeruginosa
fied as on the basis of colony morphology,
gram staining results, motility, oxidase reaction, produc- 4-1 Determination of MIC
tion of the pyocyanin pigment, nitrate reduction, the use
The MIC of MBL-positive isolates for imipenem is shown
of citrate and malonate as carbon sources, and the ability
Table 2
in ; among 22 MBL-positive strains, 8 were resis-
to grow at 5˚C and 42˚C.
tant. PCR detection for MBL showed that 8 strains were
Jundishapur J Nat Pharm Prod. 2012;7(1):23-26
Metallo-β–Lactamase Producers Among Pseudomonas Aeruginosa Isolates Kalantar E et al. 25
Figure 1. P. aeruginosa
Phenotypic Detection of MBLs by DDST Among Iso-
lates in Burn Patients at Tohid Hospital, Sanandaj, Iran
Table 1. In Vitro Antibiotic Resistance Pattern of Infection-Associated P. Figure 2. PCR for the Detection of blaVIM Gene
aeruginosa
Antibiotic Resistance, %
Table 2.
MIC of MBL positive isolates for Imipenem
Amikacin 52
Sensitive Intermediate Resistant
Gentamicin 54
Carbencillin 76 MICa, µg / ml
Ciprofloxacin 31 ≤ 0.125 08 16
Ofloxacin 82 0.25 32
Cefepime 72 0.5 64
Ceftazidime 96 01 128 ≤
Cefotaxime 88 02
Ampicillin 100 04
P. aeruginosa
ceftriaxone 89 No. of Isolates, %
Imipenem 21 02 01 02
Meropenem 14 02 01
04 03
bla bla bla
positive for VIM1(. FTighuer eo 2t)h. er genes, SIM1 and GIM1, 02 02
were not detected
03
5. Discussion 00
P. aeruginosa
Total of Isolates
Because of its broad antimicrobial spectrum and sta-
β 13 01 08
bility against most common -lactamases, imipenem
generally represents one of the last alternatives for the a Abbreviation: MIC: Minimum Inhitory Concentration
treatment of nosocomial infections caused by multi-
P. ae- P. aerugi-
drug-resistant gram-negative bacteria, particularly (18). In this study, 22% of the imipenem-resistant
ruginosa nosa
(13). However, the rapid spread of MBLs among isolates were MBL-positive, with 8% positive for VIM1,
major gram-negative pathogens, particularly P. aeru- which is by far the most prevalent MBL in Iran20. To our
ginosa
, is an emerging threat and a matter of concern knowledge, MBLs of other families like SIM have not been
P. aeruginosa
worldwide (14-16); further, MBL-carrying bacteria are detected in . The prevalence of MBL-produc-
P. aeruginosa
known to cause recalcitrant nosocomial infections (17). ing differs across Iranian studies, which
MBLs of the IMP, VIM, and SIM families are frequently de- may be because of differences in geographic regions.
P. aeruginosa
tected in imipenem-resistant in Iran (18-20). Further investigation is required to gain a better under-
P. aeruginosa
In our study, a total of 100 strains were iso- standing of the epidemiology and genetic background
P. aeruginosa
lated from hospitalized burn patients in Sanandaj, Iran, of MBL-producing . The uncontrolled spread
in 2010, and 22% of these strains were found to be MBL of MBL producers in hospitals may hamper treatment

producers, which ismuch lower than the findings from procedures and lead to increased morbidity and mortal-
the study conducted by Mihani and Khosravi in Ahvaz ity. Systems for regular screening should be established
Jundishapur J Nat Pharm Prod. 2012;7(1):23-26
26 Kalantar E et al. Metallo-β–Lactamase Producers Among Pseudomonas Aeruginosa Isolates
to control the spread of MBL producers, and effective pitals in 2003: Continued prevalence of VIM-producing pseudo-
monas spp. and increase of IMP-producing Acinetobacter spp.
infection-control programs in hospitals should be de- Diagnos Microbiol Infect Dis 50
. 2004; (1):51-8.
veloped and implemented thoroughly. The study results 7. Libisch B, Gacs M, Csiszar K, Muzslay M, Rokusz L, Fuzi M. Isola-
demonstrate the serious therapeutic threat of the spread tion of an integron-borne blaVIM-4 type metallo-beta-lactamase
of MBL-producing P. aeruginosa. This number (22% of imi- gene from a carbapenem-resistant Pseudomonas aerugi-
Antimicrob Agents Chemother
P. aeruginosa) nosa clinical isolate in Hungary. .
penem-resistant might have been higher if 48
2004; (9):3576-8.
other genes were included. Early detection and infection 8. Peleg AY, Franklin C, Bell J, Spelman DW. Emergence of IMP-4
J Anti-
control practices are the best defense against this organ- metallo-beta-lactamase in a clinical isolate from Australia.
microb Chemother 54
. 2004; (3):699-700.
ism; therefore, systematic surveillance to detect MBL pro-
9. Hooper DC. Emerging mechanisms of fluoroquinolone resis-
ducers is necessary. Emerg Infect Dis 7
tance. . 2001; (2):337-41.
10. Richet HM, Mohammed J, McDonald LC, Jarvis WR. Building
Acknowledgments
communication networks: international network for the study
Emerg In-
and prevention of emerging antimicrobial resistance.
fect Dis 7
None declared. . 2001; (2):319-22.
11. Performance Standards for Antimicrobial Susceptibiliti Test-
Clin Lab Stand Inst
Financial Disclosure ing; Twentienth Informational Supplement. .
30
2010; (15):10-7.
12. Pitout JD, Gregson DB, Poirel L, McClure JA, Le P, Church DL. De-
None declared.
tection of Pseudomonas aeruginosa producing metallo-beta-
J Clin Microbiol
lactamases in a large centralized laboratory. .
Funding/Support 43
2005; (7):3129-35.
13. Thomson JM, Bonomo RA. The threat of antibiotic resistance in
Funding for this project was provided by Vice-chancel- Gram-negative pathogenic bacteria: beta-lactams in peril! Curr
Opin Microbiol 8
lor in Research Affairs of Kurdistan University of Medical . 2005; (5):518-24.
14. Cornaglia G, Akova M, Amicosante G, Canton R, Cauda R, Doc-
Sciences, Sanandaj, Iran.
quier JD, et al. Metallo-beta-lactamases as emerging resistance
Int J An-
References dtiemtiecrrmobi nAagnentst sin Gram29-negative pathogens: open issues.
. 2007; (4):380-8.
15. Nam Hee R, Jung Sook H, Dong Seok J, Jae Ryong K. Prevalence of
1. Sader HS, Gales AC, Pfaller MA, Mendes RE, Zoccoli C, Barth A, et β
Metallo- -lactamases in Pseudomonas aeruginosa and Acineto-
al. Pathogen frequency and resistance patterns in Brazilian hos- Korean J Clin Microbiol 13
bacter baumannii. Korean. . 2010; (4):169-
pitals: summary of results from three years of the SENTRY Anti-
Braz J Infect Dis 5 72.
microbial Surveillance Program. . 2001; (4):200-
16. Saderi H, Karimi Z, Owlia P, Bahar MA, Rad SMBA. Phenotypic
14.
Detection of Metallo-beta-lactamase Producing Pseudomonas
2. Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable Iran J Patho
J Hosp Infect 73 aeruginosa Strains Isolated from Burned Patients. .
and ever-present adversary. . 2009; (4):338-44. 3
β 2008; (1):20-4.
3. Khosravi Y, Tee Tay S, Vadivelu J. Metallo- -lactamase–producing
17. Sarkar B, Biswas D, Prasad R, Sharma JP. A clinicomicrobiologi-
imipenem-resistant Pseudomonas aeruginosa clinical isolates
cal study on the importance of pseudomonas in nosocomially
in a university teaching hospital in Malaysia: detection of IMP-7
Diagnos Micro- infected ICU patients, with special reference to metallo beta1-
and first identification of IMP-4, VIM-2, and VIM-11. Indian J Pathol Microbiol 49
biol Infect Dis 67 lactamase production. . 2006; (1):44-8.
. 2010; (3):294-6.
18. Mihani F, Khosravi A. Isolation Of Pseudomonas Aeruginosa
4. Wirth FW, Picoli SU, Cantarelli VV, Goncalves AL, Brust FR, Santos
Strains Producing Metallo Beta Lactamases From Infections
LM, et al. Metallo-beta-lactamase-producing Pseudomonas ae-
Braz J Infect Dis In Burned Patients And Identification Of Bla Imp And Bla Vim
ruginosa in two hospitals from southern Brazil. . Iran J Med Microbiol 1
13 Genes By Pcr. . 2007;(1):23-31.
2009; (3):170-2.
19. Rezaei H, Behzadian G, Najar S, Mostafaei M. Prevalence and de-
5. Yan JJ, Hsueh PR, Ko WC, Luh KT, Tsai SH, Wu HM, et al. Metallo- ββ
tection of metallo- -lactamase (MBL)-producing Pseudomonas
beta-lactamases in clinical Pseudomonas isolates in Taiwan and Annals of Micro-
An- aeruginosa strains from clinical isolates in Iran.
identification of VIM-3, a novel variant of the VIM-2 enzyme. biology 57
timicrob Agents Chemother 45 . 2007; (2):293-5.
. 2001; (8):2224-8.
20. Yousefi S, Farajnia S, Nahaei MR, Akhi MT, Ghotaslou R, Soroush
6. Lee K, Ha GY, Shin BM, Kim JJ, Kang JO, Jang SJ, et al. Metallo-
MH, et al. Detection of metallo-beta-lactamase-encoding genes
[beta]-lactamase-producing Gram-negative bacilli in Korean
among clinical isolates of Pseudomonas aeruginosa in north-
Nationwide Surveillance of Antimicrobial Resistance group hos- Diagn Microbiol Infect Dis 68
west of Iran. . 2010; (3):322-5.
Jundishapur J Nat Pharm Prod. 2012;7(1):23-26