Guiana Extended Spectrum Ges Enzymes

Published Date: 02 Nov 2017

Background:

Guiana extended-spectrum (GES) enzymes have been described occurring in P.

aeruginosa worldwide. This enzyme hydrolyses expanded-spectrum cephalosporins

and, to a minor extent, imipenem. The aim of this study was to detect the spread of the

GES-2 ß-lactamase gene among ESBLs producing P. aeruginosa isolates in a Kashan

teaching hospital in Iran.

Materials and Methods:

The detection of ß-lactamase gene blaGES-2 in eight ESBL producing isolates of P.

aeruginosa collected from 100 clinical and environmental specimens were performed

by PCR method. The susceptibility of the isolates to eight antibacterial agents

determined by Disk diffusion method. ESBL production of the isolates was detected

by double disk synergy test. DNA sequencing and aligning with reference sequence

using BLAST and CLUSTAL W for selected PCR products of the blaGES-2 gene were

performed.

Results:

Eight of the isolates were found to be ESBL positive. The highest resistance rate was

detected for piperacillin (75%), while the lowest susceptibility rate was for

ciprofloxacin (12.5%). It was detected that 50% of the isolates were imipenem-

susceptible and MDR. The blaGES-2 gene was detected in all ESBL producing isolate.

The sequencing of both forward and reverse strands of the blaGES-2 gene were

identified by BLASTn search as the ATP-binding cassette (ABC) transporter

permease, partial cds, clone G-1 Pseudomonas aeruginosa (Accession no.

AB591379.1).

Conclusions:

To our knowledge, this is the first report of the presence of blaGES-2 gene of ESBLs

producing P. aeruginosa from teaching hospital in Iran. Fifty percent of the blaGES-2

positive P. aeruginosa isolates were multi-drug resistance (MDR).

Keywords: Pseudomonas aeruginosa, extended-spectrum ß-lactamases, blaGES-2 gene,multidrug resistant

Background

Clavulanic acid inhibited extended-spectrum ß-lactamases (ESBLs) have been

described primarily in Enterobacteriaceae then in P. aeruginosa [1, 2]. Usually,

ESBL enzymes have been products of TEM and SHV parental enzymes. PER, GES,

IBC or certain OXA type enzymes, have also been explained in some of countries [3].

Guiana Extended-Spectrum (GES), the new class of ESBLs, was reported in a clinical

isolate of K. pneumoniae from French Guiana in 2000[4]. The identification of GES-2

is an interesting research progress in ESBLs in P. aeruginosa [5]. The GES-2 ß-

lactamase is a class A carbapenemase, the emergence of which in clinically

considerable bacterial pathogens is an alarming advance as the enzyme confers

resistance to carbapenem antibiotics [6]. The integron genetic framework of GES-2 is

the essential factor that emerge resistance to broad-spectrum ß-lactam antibiotics and

other dissimilar classes of antimicrobials [5]. Outcome of introducing such

characteristics produce a huge challenge to effective treatment and control of these

isolates on health system. [7]. blaGES-2 is put up a gene cassette in a class 1 integron.

GES-2 was at first reported in a MDR clinical isolate of P. aeruginosa originating

from a university hospital in South Africa [8]. GES-2 was associated with an outbreak

occurring in the same hospital in 2000 [9]. Inhibitory activity to imipenem is extended

by substitution of glycine to asparagine in GES-2[10, 11]. Rapid recognition of resistant strains and detection of resistance genes is

necessary to success in antimicrobial therapy and infection control policy. The aims

of this study were to examine the presence of ß-lactamase gene blaGES-2 in ESBL

producing isolates of P. aeruginosa, DNA sequencing for selected PCR products of

the blaGES-2 gene, sequence comparing and aligning with reference sequence and to

evaluate their resistance to eight commonly used antibiotics.

Materials and Methods

A total of eight ESBL producing isolates of P. aeruginosa collected during February

2010 to September 2011 from Beheshti hospital in Kashan, Iran. The strains were

isolated from blood (1), trachea (2), urine (2), gastrointestinal fluid (1) and

bronchoscopy fluid (1) and wet environment of hospital (1). The susceptiblities of the

bacterial isolates to eight antimicrobial agents imipenem (10µg), ciprofloxacin (5µg),

ceftazidime (30µg), ceftriaxone (30µg), cefotaxime (30µg), aztreonam (30µg),

piperacillin (100µg) and gentamicin (10µg) [Mast Group Ltd., Merseyside, U.K.]

were determined by the disk diffusion method according to the criteria published by

the Clinical and Laboratory Standards Institute (CLSI) [12]. Quality control in

antibiotic susceptibility determined by P. aeruginosa ATCC 27853. Double disk

synergy test used for ESBL production as described by Jarlier [1]. The detection of ß-

lactamase gene blaGES-2 was performed under standard PCR conditions using published set of primers (Table 1). For PCR, a 1:10 dilution of 24 hours culture was boiled for 10 min. Then amplification was performed with 1:10 of this dilution as the DNA template. PCR settings involved 30 cycles of amplification under the following

conditions: Denaturation at 95° C for 30 sec, annealing at 50° C for 60 sec and then at

72° C for 60 sec, and cycling was followed by a final extension at 72°C for 5 min.

The PCR products were analyzed by electrophoresis on a 1.5% agarose gel. After

electrophoresis, gel was stained with ethidium bromide and photographed under a UV

trans-illuminator (Ingenius, Syngene, UK). A 100-bp DNA (Bioneer, Korea) ladder

was used as a molecular size marker. DNA sequencing for selected PCR products of

the blaGES-2 gene were performed for identification of detected bla gene using primers

as shown in Table1. The PCR products of above gene subjected to direct sequencing

of both the strands performed using an automated sequencer (ABI system, 3730XL)

by Macrogen Company in Korea. The nucleotide sequences were analyzed with

Chromas LITE version 2.01 software. Sequences were compared and aligned with

reference sequence using BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and

CLUSTAL W http://www.ebi.ac.uk/clustalw).

The nucleotide sequences of blaGES-2 gene and partial gene encoding ABC transporter

permease(G-1) were deposited in GenBank through DNA Data Bank of Japan (DDBJ

), and the accession number assigned were AF326355.1 and AB591379.1

respectively.

Results

The prevalence rate of ESBLs was 8%. The resistance pattern of eight ESBL

producing P. aeruginosa isolates in our study showed the wonderful high resistance

rate to piperacillin (75%) and all extended-spectrum cephalosporins (62.5%). The

isolated ESBL producing P. aeruginosa were more sensitive to imipenem, gentamicin

and ciprofloxacin than other tested antibiotics (Table 2). From the eight isolates, 4

(50%) were resistant to at least three classes of antibiotics and classified as multi-drug

resistance (MDR). By using primer pair blaGES-2 , visible bands near the location of

expected size were observed in eight isolates. In order to increase the accuracy of the

results, the sequencing of both forward and reverse strands of the blaGES-2 gene were

carried out twice (Fig. 1). The sequence alignment with reference sequence blaGES-2

(Accession no. AF326355.1) revealed no similarity by BLASTn search and

CLUSTAL W and were identified by BLASTn search as the ATP-binding cassette

(ABC) transporter permease, partial cds, clone G-1 Pseudomonas aeruginosa

(Accession no. AB591379.1) with 84% coverage and 100% maximum identity.

Discussion

In our study, the blaGES-2 gene was detected in all ESBL producing isolates. GES ß-

lactamase genes are placed on broad-host-range conjugative plasmids, are element of

gene cassettes in class 1 integrons, and are found in gram-negative isolates, they may

have extend worldwide[13]. GES-2 ß-lactamase is the fourth example of a non-TEM-,

non-SHV-type ESBL in P. aeruginosa after PER-1, VEB-1, and OXA-18 [8]. GES-

type ß-lactamase consist of 15 members (GES-1 to GES-15), isolated from various

Gram-negative bacteria in Europe, Asia, Africa, and the Americas[13]. Although

some GES ß-lactamases produce antibiotic resistance profiles like those of classical

extended spectrum enzymes, some GES variants (GES-2, -4, -5, and -6) also confer

reduced susceptibility to imipenem [13]. GES ß-lactamases are found in French

Guiana, Greece, and South Africa [14]. blaGES-1 has also been found in a P.

aeruginosa strain isolated in French medical center and from a 63-year-old female

who had a hysterectomy (São Paulo, Brazil) [15]. GES-2 was identified from a P.

aeruginosa isolate in South Africa and later from 8 more strains involved in the

outbreak. GES-2 ß-lactamase may supply partly to the decreased susceptibility of P.

aeruginosa to imipenem [8, 16, 17]. The GES-2 ß-lactamase belongs to class A

carbapenemase, the emergence of which in P. aeruginosa is an alarm as the enzyme

confers resistance to carbapenem antibiotics. Tazobactam is a clinically used inhibitor

of class A ß-lactamases, which inhibits the GES-2 enzyme efficiently, returning

susceptibility to ß-lactam antibiotics.

Our study showed the sequencing of the blaGES-2 gene that was identified by BLASTn,

was matched as the ATP-binding cassette (ABC) transporter permease, partial cds,

clone G-1 P. aeruginosa (Accession no. AB591379.1). Likely, same results were

obtained by Hirakawa, et al. [18]. Resistance in P. aeruginosa may be mediated via

several distinct mechanisms including the production of ß- lactamases, efflux pumps,

and target-site or outer membrane modifications [19]. Efflux pump systems are

emerging as extremely important causes of multidrug resistance in P. aeruginosa.

ABC transporters are major efflux pump protein families that mediate resistance to

antibiotics and are known to play a crucial role in the development of MDR [20, 21].

In general, have been found in both prokaryotic and eukaryotic systems and are

responsible for the import and export of various proteins, peptides, polysaccharides,

and drugs and utilize ATP hydrolysis to drive the export of substrates [21].

The detection of the extended-spectrum ß-lactamase (ESBL) GES-2 from P.

aeruginosa in the 7 hospitalized patients and one environment setting, it became clear

that they were established in the our teaching hospital. P. aeruginosa producing GES-

2 tendency to colonize and infect mostly debilitated patients, considerably increasing

both their lengths of stay in the hospital and the cost of treatment. The integron

genetic structures that support GES-2, not only present resistance to broad-spectrum

ß-lactam antibiotics but also to dissimilar classes of antimicrobials, making these

isolates very difficult to treat and control successfully.

In our study, prevalence of MDR P. aeruginosa isolates, was 50% which lower than

value reported in study conducted by Aggarwal et al. (100%) [22].

Carbapenems were subsequently introduced into the clinic as last resort antibiotics

due to their high potency and exceptional broad spectrum of antimicrobial activity

that includes both Gram-negative and Gram-positive aerobic and anaerobic bacteria.

New types of class A ß-lactamases, the carbapenemases are capable of producing

resistance to a wide variety of ß-lactam antibiotics, including carbapenems[6]. In this

study, notable resistance (50%) to P. aeruginosa was observed against imipenem,

while other studies reported that more than 80% of ESBL producing isolates were

sensitive to imipenem [23, 24, 25]. Our study is the first to demonstrate the presence

of blaGES-2 in P. aeruginosa in Iran. ESBLs are an uncommon but significant problem

in our hospital. GES-2 was found in all 0f ESBL-positive isolates and half of them

were MDR.

Acknowledgements

The manuscript based on the thesis of MSc degree and was supported by Kashan

University of Medical Sciences Research fund.

Ethical Consideration

All Ethical issues (such as informed consent, conflict of interest, plagiarism,

misconduct, co-authorship, double submission) have been considered carefully .