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Molecular Detection of blaVEB-1 Beta-Lactamase Encoding Gene Among Extended Spectrum B-Lactamase Positive Wound Isolates of Pseudomonas aeruginosa


1 Department of Microbiology, School of Paramedical Sciences, Ilam University of Medical Sciences, Ilam, IR Iran
2 Department of Microbiology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, IR Iran
3 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran
4 Research Center of Pediatric Infectious Diseases, Iran University of Medical Sciences, Tehran, IR Iran
*Corresponding author: Elham Davodian, Department of Microbiology, School of Paramedical Sciences, Ilam University of Medical Sciences, P. O. Box: 6931873911, Ilam, IR Iran. Tel: +98-8413383711, Fax: +98-8413383711, E-mail: Elhamdavudian@yahoo.com.
Archives of Pediatric Infectious Diseases. 2015 October; 3(4): e26362 , DOI: 10.5812/pedinfect.26362
Article Type: Research Article; Received: Dec 23, 2014; Revised: Jun 24, 2015; Accepted: Aug 9, 2015; epub: Oct 12, 2015; ppub: Oct 2015

Abstract


Background: Pseudomonas aeruginosa is considered as a leading cause of nosocomial infections. Burn and wound infections are mainly caused by multidrug-resistant P. aeruginosa isolates. Drug resistance frequently occurs among nosocomial isolates and can usually resist a myriad of antibiotics such as novel β-lactam antibiotics. Detection of multidrug-resistant isolates could assist better drug administration.

Objectives: The aim of this study was to detect Extended Spectrum Beta-Lactamases (ESBL) positive wound isolates and the genes encoding blaVEB-1 ESBL among wound isolates of P. aeruginosa.

Materials and Methods: A total of 89 wound isolates of P. aeruginosa were collected from patients (47% (n = 42) were male and 53% (n = 47) were female) at six Iranian hospitals between years 2009 and 2011. Antibiotic susceptibility and phenotypic ESBL production tests were conducted. The combined disk was used to determine ESBLs production. The blaVEB-1 gene was detected with the polymerase chain reaction (PCR).

Results: The majority of the wound isolates were resistant to augmentin (90%, n = 80) and cefpodoxime (87.6%, n = 78). However, the majority was susceptible to imipenem and meropenem. Fifty-eight (42%) wound isolates were ESBL positive. The antibiotic resistance amongst ESBL positive isolates was relatively higher than ESBL negative isolates. Twenty-three (40%) ESBL-positive isolates amplified the blaVEB-1 gene.

Conclusions: More than behalf of the wound isolates were ESBL positive, and the presence of blaVEB-1 was determined in less than half of these isolates. Fortunately, resistance to imipenem and meropenem was low.

Keywords: Extended Spectrum Beta Lactamases; Wound Samples; bla VEB-1; Pseudomonase aeruginosa

1. Background


Pseudomonas aeruginosa (P. aeruginosa) isolates are known as potential opportunistic organisms, frequently involved in infections of immune suppressed or hospitalized patients, and also cause outbreaks of hospital-acquired infections. These strains are inherently resistant to an extended spectrum of antibiotics, including novel β-lactam agents, and thereby can culminate in high morbidity and mortality rates (1). Useful antibiotics include extended spectrum beta-lactamases (ESBL) and carbapenems, though multidrug-resistant isolates have emerged in hospital settings (2). Of several primary antibiotic resistant mechanisms, the down-regulation of membrane porins (OprD), in addition to increase in the expression of multidrug efflux pumps (MexAB-OprM) help intrinsic drug resistance (3). Novel beta-lactamases, including AmpC, extended spectrum beta-lactamases (ESBLs) and likewise several metallo beta-lactamases (MBLs) have emerged around the world as genetic encoding reservoirs responsible for the antimicrobial resistance among different gram-negative isolates (4). The ESBL enzymes are encoded by plasmids and integrons and were first reported in isolates of Klebsiella pneumonia, in Germany (5). In P. aeruginosa several classes of enzymes including class A ESBLs, which are comprised of blaPER-1 and blaVEB-1, and GES/IBC and BEL types have been identified; these were initially reported in Turkey, south Asia and France. These six types of ESBLs at the genetic level have a low similarity, but they are identical regarding hydrolysis profiles (6). The ESBL enzymes have been demonstrated to be the derivatives of TEM- and SHV-lactamases, with minor genetic mutation in the active site (7). These enzymes have shown resistance to extended spectrum cephalosporins. Furthermore, there have reports of non-TEM and non-SHV ESBLs in several areas (8, 9). Among several acquired beta-lactamase enzymes, the blaVEB-1 has the greatest clinical importance as it causes resistance to oxyimino beta-lactams (10). On the other hand, resistance to carbapenems in P. aeruginosa, alongside K. pneumoniae and Acinetobacter baumannii, is of high concern (11). Implication for health policy/practice/research/medical education: P. aeruginosa is a nosocomial pathogen and likewise the increasing rate of antibiotic resistance has become a great concern. Among several mechanisms of drug resistance, there are ESBL enzymes that confer the resistance to a broad range of antibiotics in beta lactam family. Detection of ESBLs and their importance and prevalence can help for better follow up of this pathogen.

2. Objectives


The aim of this study was to detect the production of ESBLs and prevalence of blaVEB-1 gene among wound isolates of P. aeruginosa.

3. Materials and Methods


A total of 89 clinical isolates of P. aeruginosa were collected from wound samples in several hospitals between years 2009 and 2011. The isolates were identified by gram staining, catalase and oxidase tests, motility on Sulfide indole motility (SIM) medium, indole production, H2S production, characteristics on the triple sugar iron (TSI) agar culture medium, methyl red (MR) test, voges proskauer (VP) medium, Simon citrate agar, oxidative/fermentative (OF) test, urea broth, growth on MacConkey agar and also on cetrimide agar media. The isolates were then subsequently stored at -70°C for future studies.


The antibiotic susceptibility test of the isolates was performed on the basis of Clinical and Laboratory Standards Institute (CLSI) guidelines. The antibiotic disks used in the present study have been depicted in Table 1.


Table 1.
Three Families of Antibiotics Used in This Study

Production of ESBLs by P. aeruginosa isolates was determined using the usual combined disk test. In the combined disk test, both ceftazidime and cefotaxime disks in the presence or absence of clavulanic acid were used on the muller hinton agar (MHA) culture Plates. A positive test was indicated when the difference in the diameter in the absence of clavulanic acid was equal or more than 5 mm when compared to the diameter in the presence of clavulanic acid.


Following the suspension of a colony of each bacterial isolate in 10 mL of Luria Bertani (LB) broth medium, and then incubation overnight at 37°C, the tubes were centrifuged for 10 minutes at 4000 rpm and the obtained precipitate was re-suspended in sterile H2O for DNA extraction. Furthermore, for DNA isolation, the boiling and DNA Extraction kit (DIAtom DNA Prep 100) methods were used.


The PCR was performed for the detection of the ESBL encoding gene of blaVEB-1 by the employment of specific primers, as shown in Table 2.


Table 2.
The Primers Used in This Study

The reaction mixture for these genes included: 10X PCR buffer = 2.5 µL, dNTP (10 Mm) = 0.75 µL, MgCl2 (50 mM) = 1.5 µL, forward primer (100 µM) = 2.5 µL, reverse primer (100 µM) = 2.5 µL, template (DNA) = 1 µL, Taq DNA polymerase (5 U/µL) = 0.2 µL, and nuclease-free H2O = 14.05 µL.


3.1. Statistical Analysis

The analysis of data was performed with application of the Student’s t-test.

4. Results


The wound isolates of P. aeruginosa were collected from six hospitals of Tehran (Shariati, burn center), Shiraz (Namazi), Ilam (Imam Khomeini), Kerman (Bahonar), Kermanshah (Imam Khomeini), and Ahvaz (Imam) city. These isolates were identified with conventional biochemical tests.


The majority of the wound isolates were susceptible to imipenem and meropenem antibiotics. However, most were resistant to augmentin disk and cefpodoxime. The difference between ESBL- and non-ESBL-producing isolates regarding resistance has been classified in Table 3.


Table 3.
The Antibiotic Susceptibility Test Pattern for the Extended Spectrum B-Lactamases Positive and Negative Wound Pseudomonas aeruginosa Isolates

Fifty-eight (56.1%) of the wound P. aeruginosa isolates were ESBL positive, among which 40% (n = 26) were isolated from males and 60% (n = 32) from females. The antibiotic resistance pattern was higher in these isolates relative to ESBL-negative isolates, although no significant difference was observed (P ≤ 0.05), as shown in Table 3. The prevalence of ESBLs in each hospital were as follows; Tehran (n = 11), Shiraz (n = 6), Ilam (n = 8), Kerman (n = 11), Kermanshah (n = 8) and Ahvaz (n = 6).


The prevalence of blaVEB-1 in each hospital was as follows; Tehran (n = 10), Shiraz (n = 2), Ilam (n = 8), Kerman (n = 3), Kermanshah (n = 5) and Ahvaz (n = 4). Isolates that contained blaVEB-1 gene also showed higher resistance to third generation antibiotics. As described previously, several ESBL positive isolates in some hospitals did not amplify the blaVEB-1 gene. Table 4 shows the association between third-generation cephalosporins resistance and extended spectrum beta-lactamase genotypes.


Table 4.
Association Between Third-Generation Cephalosporins Resistance and Extended Spectrum Beta-Lactamase Genotypesa

5. Discussion


Drug resistance is increasingly developing amongst nosocomial pathogens (13, 14). Approximately 0.3% of P. aeruginosa genes encode agents for antibiotic resistance (15). The ESBL-positive P. aeruginosa strains are resistant to the extended-spectrum cephalosporins with several estimated mechanisms (16). In this study, more than 45% of the ESBL positive wound isolates contained the blaVEB-1 gene, suggesting that several other mechanisms can also interfere in decreased resistance to third generation cepgalosporins; such as reducing the levels of antibiotics accumulated in bacteria or increasing the expression of efflux pumps that are important in gram negative strains. In the present study, the majority of the wound P. aeruginosa isolates were resistant to disks of cefpodoxime and augmentin/co-amoxi clav. We observed that most of our wound isolates were sensitive to imipenem and meropenem. Moreover, about 40% of the isolates were resistant to cefpodoxime, aztreonam, ceftriaxone and cefotaxime. The combined disk is routinely used for detection of phenotypic positive ESBLs with use a third generation cephalosporin with or without inhibitory clavulanate (17). However, resistance to the inhibitor indicates the possible presence of AmpC or other consistent enzymes (18). Several previous studies that aimed to detect ESBLs have demonstrated a high level of resistance among P. aeruginosa isolates to antibiotics (19). In this study, more than half of the wound ESBL positive isolates could amplify blaVEB-1 (47%). As mentioned above, the antibiotic resistance pattern was considerably at a higher level in ESBL positive isolates (not significant). Interestingly, the blaVEB-1 was detected in isolates that were resistant to all the used third generation cephalosporins. There are limited results regarding the prevalence of the blaVEB-1 gene in Middle Eastern countries. Amongst the results from our country, 24% of ESBL positive isolates in the study of Shacheraghi et al. contained this gene (20). Furthermore, in Tehran, Mirsalehian demonstrated that 49.25% and 31.34% of ESBL positive isolates collected from burn patients amplified blaPER-1 and blaVEB-1 genes, respectively (21). However, in Korea, none of the P. aeruginosa isolates could amplify the blaVEB-1 gene (10). Although we detected this gene at a low prevalence, because of its plasmid borne nature, there is a possibility of rapid transmission amongst gram-negative bacteria. Fortunately, carbapenem resistance was not high, as found by the study of Mirsalehian (21). However, another study exhibited that 95% of ESBL positive isolates of P. aeruginosa were resistant to imipenem and meropenem; such findings may be warning of a crisis, as these drugs are the best choices for ESBL positive isolates. In an Iranian study, conducted in the Semnan province during 2010, 88% of gram negative isolates harbored ESBLs (22). However, Aminzadeh in a study conducted in Tehran during 2011, determined that 13.7% of enteric pathogens (a total of 292 species) were ESBL positive (23). Khosravi in 2012 investigated several isolates of Klebsiella pneumonia and found that 47.27% were ESBL positive containing TEM-1 (34.61%), SHV-1 (46.15%) and CTX-M-1 (26.92%) genes (24). In the research of Fazeli, 71% of K. pneumonia isolates were ESBL positive (25). In the study of Kapur from India, 61% of urinary tract pathogens were ESBL positive (26). For resistant isolates combination therapy (usually including a class of β-lactam and an aminoglycoside) is recommended that would contribute to the curing of pseudomonal infections (27). Less than half of our wound isolates of P. aeruginosa produced ESBLs among which an approximate half could amplify the blaVEB-1 gene. These isolates showed a higher drug resistance compared to ESBL negative strains. On the other hand, the resistance to carbapenems was low.

Acknowledgments

This study was supported by Ilam University of Medical Sciences.

Footnotes

Authors’ Contributions: Elham Davodian designed the experiments, Nourkhoda Sadeghifard advised the study, Abdolmajid Ghasemian helped with the data analysis and writing of the manuscript, and Samileh Noorbakhsh helped with the study in the laboratory process.

References


  • 1. Basak S, Khodke M, Bose S, Mallick SK. Inducible Amp C beta-lactamase producing Pseudomonas aeruginosa isolated in a rural hospital of central India. J Clin Diagn Res. 2009;3:19-7.
  • 2. Kohlenberg A, Weitzel-Kage D, van der Linden P, Sohr D, Vogeler S, Kola A, et al. Outbreak of carbapenem-resistant Pseudomonas aeruginosa infection in a surgical intensive care unit. J Hosp Infect. 2010;74(4):350-7. [DOI] [PubMed]
  • 3. El Amin N, Giske CG, Jalal S, Keijser B, Kronvall G, Wretlind B. Carbapenem resistance mechanisms in Pseudomonas aeruginosa: alterations of porin OprD and efflux proteins do not fully explain resistance patterns observed in clinical isolates. APMIS. 2005;113(3):187-96. [DOI] [PubMed]
  • 4. Gupta V, Garg R, Garg S, Chander J, Attri AK. Coexistence of Extended Spectrum Beta-Lactamases, AmpC Beta-Lactamases and Metallo-Beta-Lactamases in Acinetobacter baumannii from burns patients: a report from a tertiary care centre of India. Ann Burns Fire Disasters. 2013;26(4):189-92. [PubMed]
  • 5. Qi Y, Wei Z, Ji S, Du X, Shen P, Yu Y. ST11, the dominant clone of KPC-producing Klebsiella pneumoniae in China. J Antimicrob Chemother. 2011;66(2):307-12. [DOI] [PubMed]
  • 6. Strateva T, Yordanov D. Pseudomonas aeruginosa - a phenomenon of bacterial resistance. J Med Microbiol. 2009;58(Pt 9):1133-48. [DOI] [PubMed]
  • 7. Mansour W, Dahmen S, Poirel L, Charfi K, Bettaieb D, Boujaafar N, et al. Emergence of SHV-2a extended-spectrum beta-lactamases in clinical isolates of Pseudomonas aeruginosa in a university hospital in Tunisia. Microb Drug Resist. 2009;15(4):295-301. [DOI] [PubMed]
  • 8. Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004;48(1):1-14. [PubMed]
  • 9. Luzzaro F, Mezzatesta M, Mugnaioli C, Perilli M, Stefani S, Amicosante G, et al. Trends in production of extended-spectrum beta-lactamases among enterobacteria of medical interest: report of the second Italian nationwide survey. J Clin Microbiol. 2006;44(5):1659-64. [DOI] [PubMed]
  • 10. Shahcheraghi F, Nikbin VS, Feizabadi MM. Prevalence of ESBLs genes among multidrug-resistant isolates of Pseudomonas aeruginosa isolated from patients in Tehran. Microb Drug Resist. 2009;15(1):37-9. [DOI] [PubMed]
  • 11. Papp-Wallace KM, Endimiani A, Taracila MA, Bonomo RA. Carbapenems: past, present, and future. Antimicrob Agents Chemother. 2011;55(11):4943-60. [DOI] [PubMed]
  • 12. Poirel L, Menuteau O, Agoli N, Cattoen C, Nordmann P. Outbreak of extended-spectrum beta-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J Clin Microbiol. 2003;41(8):3542-7. [PubMed]
  • 13. Ghasemian A, Peerayeh SN, Bakhshi B, Mirzaee M. Detection of accessory gene regulator groups genes and cassette chromosome mec types among Staphylococcus aureus isolated from intensive care unit patients. Asian Pacific J Trop Dis. 2015;5(2):153-7. [DOI]
  • 14. Ghasemian A, Najar Peerayeh S, Bakhshi B, Mirzaee M. Accessory Gene Regulator Specificity Groups Among Staphylococcus aureus Isolated From Hospitalized Children. Arch Pediatr Infect Dis. 2014;2(2):eee16096. [DOI]
  • 15. Mesaros N, Nordmann P, Plesiat P, Roussel-Delvallez M, Van Eldere J, Glupczynski Y, et al. Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect. 2007;13(6):560-78. [DOI] [PubMed]
  • 16. Hosseini-Mazinani SM, Eftekhar F, Milani M, Ghandili S. Characterization of beta-lactamases from urinary isolates of Escherichia coli in Tehran. Iran Biomed J. 2007;11(2):95-9. [PubMed]
  • 17. Garrec H, Drieux-Rouzet L, Golmard JL, Jarlier V, Robert J. Comparison of nine phenotypic methods for detection of extended-spectrum beta-lactamase production by Enterobacteriaceae. J Clin Microbiol. 2011;49(3):1048-57. [DOI] [PubMed]
  • 18. Wolter DJ, Schmidtke AJ, Hanson ND, Lister PD. Increased expression of ampC in Pseudomonas aeruginosa mutants selected with ciprofloxacin. Antimicrob Agents Chemother. 2007;51(8):2997-3000. [DOI] [PubMed]
  • 19. Salimi F, Eftekhar F. Coexistence of AmpC and Extended-Spectrum β-lactamases in Metallo-β-Lactamase Producing Pseudomonas aeruginosa Burn Isolates in Tehran. Jundishapure J Microbiol. 2013;6(8):eee7178 [DOI]
  • 20. Shacheraghi F, Shakibaie MR, Noveiri H. Molecular Identification of ESBL genes blaGES-1, blaVEB-1, blaCTX-M blaOXA-1, blaOXA-4, blaOXA-10 and blaPER-1 in Pseudomonas aeruginosa strains isolated from burn patients by PCR, RFLP and sequencing techniques. Int J Biol life Sci. 2010;3(6):138-42.
  • 21. Mirsalehian A, Feizabadi M, Nakhjavani FA, Jabalameli F, Goli H, Kalantari N. Detection of VEB-1, OXA-10 and PER-1 genotypes in extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa strains isolated from burn patients. Burns. 2010;36(1):70-4. [DOI] [PubMed]
  • 22. Irajian G, Jazayeri Moghadas A. Frequency of extended-spectrum beta lactamase positive and multidrug resistance pattern in Gram-negative urinary isolates, Semnan, Iran. Jundishapur J Microbiol. 2010;3(3):107-13.
  • 23. Aminzadeh Z, Yadegarynia D, Fatemi A, Azad Armaki S, Aslanbeygi B. Prevalence and Antimicrobial Susceptibility Pattern of Extended Spectrum Beta Lactamase (ESBL) and non-ESBL Producing Enteric Gram-Negative Bacteria and Activity of Nitrofurantoin in the era of ESBL. Jundishapur J Microbiol. 2013;6(7) [DOI]
  • 24. Khosravi AD, Hoveizavi H, Mehdinejad M. Prevalence of Klebsiella pneumoniae Encoding Genes for Ctx-M-1, Tem-1 and Shv-1 Extended-Spectrum Beta Lactamases (ESBL) Enzymes in Clinical Specimens. Jundishapur J Microbiol. 2013;6(10):eee8256 [DOI]
  • 25. Fazeli H, Dolatabadi RK, Taraghian A, Nasr Isfahani B, Moghim S, Norouzi M. Carbapenem Resistance Pattern of Multiple Drug-Resistantand Extended-Spectrum Beta-Lactamase-Positive Klebsiella pneumonia in Isfahan. Int J Enteric Pathog. 2014;2(4):eee21495
  • 26. Kapur A, John AS. Prevalence of Extended-Spectrum Beta-Lactamase-Producing Pathogens From Urinary Tract Infected Samples and Their Sensitivity Pattern Against. Int J Infect. 2015;2(1):eee22664
  • 27. Hill D, Rose B, Pajkos A, Robinson M, Bye P, Bell S, et al. Antibiotic susceptabilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. J Clin Microbiol. 2005;43(10):5085-90. [DOI] [PubMed]

Table 1.

Three Families of Antibiotics Used in This Study

Antibiotic Family Disks and Concentrations
Beta-lactams, µg Aztreonam (30), piperacillin (100), carbenicillin (100), meropenem (10), netilmicin (30), ticarcillin (75), piperacillin-tazobactam (110), cefoperazone (75), augmentin (30), cefotaxime (30), imipenem (10), cefpodoxime (10), ceftriaxone (30), ceftazidime (30) and cefepime (30)
Fluoroquinolones, µg Ofloxacin (5), ciprofloxacin (5), levofloxacin (5)
Aminoglycosides, µg Amikacin (30), tobramycin (10), gentamicin (120)

Table 2.

The Primers Used in This Study

Primer Sequence 5 to 3 Product Size Reference
bla VEB-1 699 (12)
F: AATGGCAATCAGCGCTTC
R: GCGCGACTGTGATGTATA

Table 3.

The Antibiotic Susceptibility Test Pattern for the Extended Spectrum B-Lactamases Positive and Negative Wound Pseudomonas aeruginosa Isolates

Disks/Isolates ESBL-Positive (Resistance %), n = 35 ESBL-Negative (Resistance %), n = 54
Augmentin 100 92
Cefepime 96 68
Ceftazidime 89 66
Cefpodoxime 86 54
Carbenicillin 87 63
Ceftriaxone 99 63
Piperacillin 88 46
Aztreonam 86 62
Cefoperazone 89 46
Cefotaxime 98 67
Ticarcillin 68 56
Imipenem 22 17
Meropenem 23 18
Ciprofloxacin 89 65
Levofloxacin 78 44
Ofloxacin 76 55
Netilmicin 63 45
Amikacin 82 42
Gentamicin 68 43
Tobramycin 68 46
Piperacillin 56 37

Table 4.

Association Between Third-Generation Cephalosporins Resistance and Extended Spectrum Beta-Lactamase Genotypesa

Third Generation Cephalosporins Resistant Isolates blaVEB-1, %
CTX 80
CAZ 89.3
CPM 78
CRO 78.4
CTX, CAZ, CRO, CPM 97.6
a Abbreviations: CTX: cefotaxime, CAZ: ceftazidime, CPM: cefpodoxime, CRO: ceftriaxone.