Prevalence of Metallo-β-lactamases Encoding Genes Among <i>Pseudomonas aeruginosa</i> Strains Isolated From the Bedridden Patients in the Intensive Care Units

Marzieh Safari; Mohammad Yousef Alikhani; Mohammad Reza Arabestani; Reza Kamali Kakhki; Rasool Jafari

doi:10.17795/ajcmi-19216

Prevalence of Metallo-β-lactamases Encoding Genes Among Pseudomonas aeruginosa Strains Isolated From the Bedridden Patients in the Intensive Care Units

Marzieh Safari ¹ , Mohammad Yousef Alikhani ^{1 , 2 , *} , Mohammad Reza Arabestani ^{1 , 2} , Reza Kamali Kakhki ¹ and Rasool Jafari ³

Authors Information

¹ Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, IR Iran

² Brucellosis Research Center, Hamadan University of Medical Sciences, Hamadan, IR Iran

³ Department of Parasitology and Mycology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran

Article Notes and Dates

Avicenna Journal of Clinical Microbiology and Infection: May 2014, 1 (1); e19216.Published Online: April 28, 2014

Article Type: Research Article; Received : February 14, 2014; Revised : March 1, 2014; Accepted : March 9, 2014
DOI: http://dx.doi.org/10.17795/ajcmi-19216

To Cite : Safari M, Alikhani M Y, Arabestani M R, Kamali Kakhki R, Jafari R. et al. Prevalence of Metallo-β-lactamases Encoding Genes Among Pseudomonas aeruginosa Strains Isolated From the Bedridden Patients in the Intensive Care Units, Avicenna J Clin Microb Infec. 2014 ;1(1):e19216. doi: 10.17795/ajcmi-19216.

Abstract

Copyright: Copyright © 0, Avicenna Journal of Clinical Microbiology and Infection. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Infectious diseases have been a prominent cause of mortalities in the whole human history (1). One of the major causes of nosocomial infections is Pseudomonas aeruginosa, which is causing almost 9% to 10% of hospital infections. In addition, it causes chronic lung infections in patients with cystic fibrosis. The antibiotic resistance is one of the reasons that makes P. aeruginosa an outstanding pathogen (2). Nowadays drug resistance is a general problem in managing infectious diseases; this problem among the gram-positive bacteria includes vancomycin-resistant Enterococcus species, methicillin-resistant Staphylococcus aureus, and multidrug-resistant Streptococcus pneumonia. The most important multidrug-resistant gram-negative bacteria are P. aeruginosa, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumonia (1). The drug resistance mechanisms in P. aeruginosa are uptake limitation through the outer membrane, energy-dependent efflux, and β-lactamase (3). Carbapenems (meropenem and imipenem) are effective against P. aeruginosa (4). Recent data suggest that the resistance to carbapenems is increasing among isolates of P. aeruginosa (5). All carbapenems are hydrolyzing by metallo-β-lactamases (MBLs) that are produced by the bacteria and make it resistant to the meropenem and imipenem. The presence of MBL-producing strains makes treatment difficult and sometimes ineffective (5, 6). The carbapenems-resistant agents are important not only in treatment failure, but also in horizontal transmission of resistance genes to the other bacteria (5, 7, 8). There are evidences showing that MBL producing P. aeruginosa strains casus high mortality (9). MBLs are produced by a group of genes from which IMP, VIM, SPM, SIM, and GIM have been identified as the source of the resistance to carbapenems (10, 11).

2. Objectives

3. Materials and Methods

3.1. Sampling and Bacterial Isolation

In a cross-sectional study, 100 P. aeruginosa isolates were collected from clinical specimens of patients in ICU wards of three educational hospitals of Hamadan City (Januvary 2012 to May 2012). These isolates were obtained from culture of specimens from tracheal aspirate, blood, urine, sputum, wound, feces, and cornea of the patients. The Isolates were identified by biochemical tests (12). The isolates were identified as P. aeruginosa if they had following characteristics: gram-negative bacilli, citrate positive, non-fermentative, TSI Alk/Alk, motile, H₂S negative, urease negative, oxidase positive, and catalase positive. The confirmed isolates kept preserved at -70°C.

3.2. Antibiogram

Susceptibility of the isolates against 18 antibiotics (Mast Co., UK) was assessed by Kirby-Bauer disc diffusion method (12, 13) and the procedure has been done according to the manufacturer’s instruction. Briefly, 1.5 × 10⁸ CFU, equivalent to No. 0.5 McFarland Turbidity Standard, was transferred to Muller-Hinton agar (Merck, Germany); antibiogram discs containing imipenem 10 µg, carbenicillin 100 µg, gentamicin 10 µg, aztreonam 30 µg, cefepime 30 µg, ceftazidime 30 µg, amikacin 30 µg, ciprofloxacin 5 µg, tigecycline 15 µg, doxycycline 30 µg, meropenem 10 µg, levofloxacin 5 µg, piperacillin 100 µg, cefotaxime 30 µg, tobramycin 10 µg, colistin sulphate 10 µg, ampicillin/sulbactam 20 µg, and piperacillin/tazobactam 110 µg were placed on the medium. Then it was incubated at 35°C for 18 hours. Results were interpreted using Clinical and Laboratory Standards Institute (CLSI) criteria (14) and reported as resistant, intermediate, and sensitive. The P. aeruginosa ATCC 27853 strain was used as quality control strain in susceptibility testing.

3.3. Minimal Inhibitory Concentration

Minimal inhibitory concentration (MIC) was determined using imipenem containing E-test strips (Lioflichem® Italy) according to the manufacturer’s instruction of the. In brief, , 1.5 × 10⁸ CFU, equivalent to McFarland Turbidity Standard No. 0.5, were transferred to Muller-Hinton agar (Merck, Germany) by sterile swab. A next, E-test strip was placed on the cultured medium and then was incubated at 35°C for 18 hours. Results were interpreted according to the CLSI criteria. The intermediate level of antibiotic susceptibility was considered resistance (13, 15).

3.4. Detection of VIM and IMP Family Genes by PCR

Single PCR was performed for amplification of P. aeruginosaVIM and IMP genes, using pair of blaVIM, IMP primers (Bioneer® Korea); VIM-F 5´-GATGGTGTTTGGTCGCATA-3´ and VIM-R 5´-CGAATGCGCAGCACCAG-3´; IMP-F 5´-GGAATAGAGTGGCTTAAYTCTC-3´and IMP-R 5´-CCAAACYACTASGTTATCT-3´. Amplification procedure was performed with 25 µL of master mix containing 2.5 µL of 10X PCR buffer with MgCl₂, 2.5 µL of dNTPs MIX (2 Mm), 1 µL of 10 pM from each forward and reveres primers, 0.2 µL of Taq polymerase 5 U/µL, 3 µL of DNA template, and 14.8 µL of DNase/RNase-Free Distilled water.

PCR was performed in the thermal cycler using the following steps: initial DNA denaturation at 94°C for five minutes, then 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 40 seconds and extension at 72°C for 50 seconds, followed by final extension at 72°C for five minutes. Agarose gel electrophoresis of the amplified DNA with 100 bp size marker (Fermentas®, Korea) were done for two hours at 80 V in a 2% agarose gel stained with ethidium bromide to detect the specific band.

3.5. Data Analysis

Data were analyzed by SPSS v.16 using Chi-square and t-tests. P < 0.05 regarded as significant. The intermediate level of susceptibility was considered as resistance in data analysis.

4. Results

Totally, 100 P. aeruginosa isolates were identified by microbiology standard methods. The resistance rates of isolates are shown in Table 1. Highest resistance rates of isolates were observed in ampicillin/sulbactam (95%), tigecycline (76%), and cefotaxime (67%). The highest sensitivity rates were seen in colistin sulphate (96%), imipenem (76%), meropenem (76%), amikacin (77%), aztreonam (77%), and piperacillin/tazobactam (81%). Moreover, 20 and 24 out of 100 isolates were resistant to imipenem in disc agar diffusion and E-test methods, respectively, but the difference was not statistically significant (P > 0.05). The MIC to imipenem was ≥ 32 ug/mL (Table 2). IMP gene was detected in four isolates, but none of the isolates were carrying VIM gene. The frequency of VIM gene was higher in imipenem-resistant isolates in DAD method (P = 0.042) (Table 3). However, in E-test method, the frequency of VIM gene was higher in isolates that were resistant to imipenem (P = 0.047).

Table 1. Antibiotic Resistance of Pseudomonas aeruginosa Against 18 Studied Antibiotics ^a

Antibiotic	Intermediate	Sensitive	Resistant
Meropenem	2	76	22
Imipenem	4	76	20
Amikacin	4	77	19
Gentamicin	0	72	28
Aztreonam	6	77	17
Cefepime	2	76	22
Ceftazidime	2	74	24
Ciprofloxacin	1	61	38
Tigecycline	13	11	76
Doxycycline	22	41	37
Levofloxacin	2	70	28
Piperacillin	0	74	26
Tobramycin	0	73	27
Colistin sulphate	0	96	4
Ampicillin/sulbactam	1	4	95
Carbenicillin	5	66	29
Piperacillin/tazobactam	0	81	19
Cefotaxime	24	9	67

^a Data are presented in No. (%).

Table 2. Minimum Inhibitory Concentration of the Studied Isolates to Imipenem by E-Test Method a

	Resistant	Intermediate	Sensitive
Break point	≤ 16	4 < X< 16	≥ 4
MIC, µg/mL	32	12	4
Isolates, %	24	1	0
Total, %	25

^aAbbreviation; MIC, minimum inhibitory concentration.

Table 3. Presence of IMP Gene in Imipenem-Resistant and Sensitive Isolates in DAD Method

	Imipenem		Total	P Value
	Resistant	Sensitive	Total	P Value
IMP gene				0.045
Positive	3	1	4
Negative	21	75	96
Total	24	76	100

Table 4. Presence of IMP Gene in Imipenem-Resistant and Sensitive Isolates in E-Test Method

	Imipenem		Total	P Value
	Resistant	Sensitive	Total	P Value
IMP gene				0.047
Positive	3	1	4
Negative	22	75	96
Total	24	76	100

5. Discussion

According to the results of the present study, drug resistance of P. aeruginosa against most of the applicable antibiotics, especially meropenem and imipenem, were relatively low in Hamadan, Iran. There are handful of studies in the country that have shown the similar condition in Iran.

Shahcheraghi et al. in Tehran carried out a similar study and reported resistance in 12.4% of 610 isolates. Interestingly, data showed that 4% of the isolates were carrying IMP gene, which is exactly equal to our study in Hamadan area (16). If we consider their study and ours, it could be concluded that a portion of imipenem-resistant P. aeruginosa carry the IMP gene and may be the cause of resistance as our findings supported a significantly higher IMP gene frequency in the resistant isolates. In another study conducted by Khosravi et al. (17) in Ahvaz, south west of Iran, the prevalence of piperacillin-, ceftazidime-, imipenem-, and meropenem-resistant P. aeruginosa isolates were 68%, 83%, 41%, and 41%, respectively, which showed higher resistance isolates in Ahvaz in comparison to Hamadam. In contrary to what we found about VIM and IMP gene, 19.5% of their imipenem-resistant isolates were carrying only VIM gene rather than IMP, which was carried by some of our isolates. If we compare the two results, it would be clear that In Hamadan area the resistance rate was lower than Ahvaz. On the other hand, gene distribution pattern was also different (17). Saderi et al. studied the antibiotic resistance of P. aeruginosa in Tehran. They reported higher prevalence of resistant isolates than ours as 74.22%, 67.19%, 64.85%, 38.28%, and 83.6% of resistance P. aeruginosa isolates were resistant to ceftazidime, piperacillin, amikacin, imipenem and gentamycin, respectively (18). As it is clear, different areas show different resistance rate even in the same country. Interestingly, Ranjbar et al. reported lower rate of antibiotic-resistant P. aeruginosa isolates from Tehran (19) than what we found in Hamadan. If we consider the aforementioned studies and compare them to our studied region, we would come to a conclusion that the resistance rate was varying in different areas and resistance genes were different too.

Continues monitoring of resistance rate of P. aeruginosa, and other pathogens is necessary in every region of the glob to have enough data about it. Consequently, it ends in designing better and successful strategies against the problems we face about antibiotic resistance. Prevalence of drug resistance among P aeruginosa strains against three antibiotics in Hamadan city was very high and alerting, but the other antibiotics were effective enough to be used in treatment of the infection. Overall, low resistance rate in this region was probably resulted from low prevalence of MBLs producing strains. Permanent monitor of changes in P. aeruginosa resistance will help to determine national priorities for local intervention efforts.

Acknowledgements

Footnotes

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