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

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 bla_PER-1 and bla_VEB-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 bla_VEB-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.

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

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.

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 bla_VEB-1 by the employment of specific primers, as shown in Table 2.

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.

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.

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 bla_VEB-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 bla_VEB-1 gene also showed higher resistance to third generation antibiotics. As described previously, several ESBL positive isolates in some hospitals did not amplify the bla_VEB-1 gene. Table 4 shows the association between third-generation cephalosporins resistance and extended spectrum beta-lactamase genotypes.

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 bla_VEB-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 bla_VEB-1 (47%). As mentioned above, the antibiotic resistance pattern was considerably at a higher level in ESBL positive isolates (not significant). Interestingly, the bla_VEB-1 was detected in isolates that were resistant to all the used third generation cephalosporins. There are limited results regarding the prevalence of the bla_VEB-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 bla_PER-1 and bla_VEB-1 genes, respectively (21). However, in Korea, none of the P. aeruginosa isolates could amplify the bla_VEB-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 bla_VEB-1 gene. These isolates showed a higher drug resistance compared to ESBL negative strains. On the other hand, the resistance to carbapenems was low.