شیوع بتالاکتامازهای نوع CTX-M در جدایه‏‏ های E. coli با مقاومت چندگانه دارویی از شمال ایران، رشت

Introduction

β-lactamases are bacterial enzymes that have the ability to hydrolyze β-lactam antimicrobial drugs. Widespread use of antimicrobials has resulted in selection of bacteria with extended spectrum β-lactamases (ESBLs) production trait. ESBLs are able to hydrolyze early cephalosporins, peniclillins, oxyimino-thiazolyl cephalosporins (including third and fourth generation cephalosporins) and monobactams, but cannot hydrolyze carbapenems or cephamycins. They are prevented by inhibitors including tazobactam, sulbactam and clavulanic acid (1 and 2). A group of class A ESBLs is CTX-M enzymes that are quicklydispersing among Enterobacteriaceaein the world (3). These periplasmic enzymes which were encoded by plasmid give a high level of resistance to cefotaxime but show a low level of activity against ceftazidime (4). Earlier CTX-M-β-lactamases were cefotaximases, but more than 60% of CTX-M variants can hydrolyze both ceftazidime and cefotaxime nowadays (1 and 2).

Primary isolation of CTX-M-1 was done from a European patient in the late 1980s (5) and since that isolation >130 CTX-M allelic variants have been reported. These CTX-M variants have been categorized in 5 major phylogenetic groups, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, or CTX-M-25 based on their amino acid sequences (3 and 5).

Dissimilar to SHV and TEM enzymes, CTX-M enzymes have not originated from point mutations in the parent enzymes, but they have originated from the chromosomal bla genes of Kluyvera sp.followed by transmission to plasmids. Further mutations resulted in variation in the CTX-M family (1 and 2). Although many Kluyvera sp. include the bla_klu genes, they don’t produce the β-lactamases and then are susceptible to cefotaxime because of weakly expression of these genes. The existence of a strong promoter upstream of the bla gene leads to cefotaxime resistance phenotype. In clinical isolates exhibiting CTX-M phenotype, insertion sequences (IS) including a strong promoter have been reported. These insertion sequences have not been observed in the natural isolates of Kluyvera (1 and 2).

In the past decade, CTX-M enzymes were the most common ESBL enzymes in clinical Enterobacteriaceaeisolates, especially in ESBL-producing Escherichia coli in Europe, Asia, and South America (3 and 6), while SHV- and TEM-type ESBL enzymes have primarily been reported from ESBL-producing K. pneumoniae and E. coli clinical isolates in North America (6).

E. coli is an important pathogen and is the causal agent of urinary tract infections (UTIs) (7). Extended-spectrum cephalosporins are usually applied for treating upper UTIs (8). Resistance to these antibiotics has been reported increasingly among E. coli strains causing UTIs, as the result of distribution of strains producing extended-spectrum-beta-lactamases (ESBLs), particularly from the CTX-M type (9 and 10). Intestinal colonization which occurs more frequently than actual infections has fueled this increase (11).

The aim of this study was to determine the antibiotic resistance patterns and the frequency of the CTX-M β-lactamase between multi-drug resistant (MDR) E. coli isolates from northern Iranian patients with UTI.

Materials and methods

Samples and strains

Urine samples were collected from appropriate patients in early morning mid-stream using sterile, wide mouthed glass bottles with screw cap tops between May and July of 2012. Samples were maintained in an ice-box until laboratory analysis. It didn't last more than one hour between sample collection and sample analysis.

Urine samples were cultured on nutrient, blood and MacConkey agar plates and incubated at 37°C for 18 to 24 h. The usual bacteriological methods were applied for cultivation, isolation and identification of isolates from urine samples. The isolates were stored at -70 ^oC in tryptic soy broth containing 15% glycerol until processing. The isolates entitled as E1 to E33.

Antibiotic susceptibility test

Disc diffusion test was applied to identify the susceptibility of isolates to the bellow antimicrobials: piperacillin (100 μg), streptomycin (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), chloramphenicol (30), amoxicillin-clavulanic acid (20/10 μg), cefepim (30 μg), ceftriaxone (30), ceftazidime (30), imipenem (10), cephalothin (30 μg), cefotaxime (30 μg) and cefuroxime (30 μg). E. coli ATCC 25922 and ATCC 35218 were used as the reference strains to control the quality of the applied antimicrobial agents. MDR were described as resistance to three or more antimicrobials. Thirty three MDR
E. coli strains were recognized (12).

Phenotypic detection of ESBLs

DDSTs were performed by placing disks of ceftazidime, cefotaxime, and cefepime (30 μg each) at 20 or 30 mm distance (center to center) from a disk containing AMC (amoxicillin, 20 μg and clavulanic acid (CLA),10 μg). When the cephalosporin zone was expanded by the clavulanate, ESBL production was supposed. It means the zones produced by the disks with clavulante were ≥5 mm larger than those without inhibitor (12).

PCR detection of bla_CTX-M

Genomic DNA was extracted by boiling a dense suspension in sterile distilled water (no. 1 McFarland standard) and then 2–3 μL of the boiled cell suspension was applied as the DNA template in the PCR assays. PCR of bla_CTX-M genes in E. coli isolates was performed using 4 primer pairs specific to each type of CTX-M including CTX-M-1, 2, 8 and 9 for detecting 516, 779, 569 and 393 bp bands, respectively (13).

Initial denaturation was done at 94ºC for 10 min followed by 30 cycles of 1 min at 94ºC, 30 sec for annealing (according to primer type) (Table 1), 1 minute at 72ºC for elongation and final extension was conducted at 72°C for 10 min. A reagent blank was included in every PCR assay which contained all components of the reaction mixture except for the bacteria. PCR products were subjected to electrophoresis in 1% agarose gel (type II, Sigma, USA) in TBE buffer at room temperature at 100 volt (50 mA) for 1 h. DNA bands were visualized by gel staining with ethidiumbromide (0.5 mg/ml) for 30 min and then photographed (14).

Results

Antibiotic susceptibility test

The bacteria isolated from urine. The resistance patterns of the thirty-three isolates were shown in Table 2. All E. coli isolates (33 strains) identified as multi-drug resistant (MDR) and also 55% of the isolates were resistant to 3^rd and 4^th cephalosporins. All isolates displayed 100% sensitivity to piperacillin.

ESBL-producing E. coli prevalence

Screening of ESBLs by DDST indicated that eight isolates (24%) were ESBL producers. Phenotypic confirmatory disc diffusion test confirmed that 8 of the 33 isolates were ESBL producers. All the ESBL isolates were completely resistant to tetracyclines, cephalothin, cefotaxime, cefuroxime and amoxicillin-clavulanic acid, but rates of resistance was as bellow: streptomycin 25%, cefepime 62%, ceftriaxone 87%, chloramphenicol 75%, ceftazidime 50%, ciprofloxacin 50% and imipenem 62%.

CTX-M in ESBL-producing E. coli

As shown in Fig. 1 and Table 3, except for one ESBL-producing isolate that could not produce any type of the CTX-M, the other isolates had one or three bla_CTX-M genes minimally. Among the bla_CTX-M genes, CTX-M-1 and CTX-M-9 showed the highest (62.5%) and the lowest (25%) abundance, respectively.

Table 1- Primers used in this study for PCR detection of bla_CTX-M

Table 2- Percentage of susceptible (S), intermediate (I) and resistant (R) E. coli strains from UTIs

* According to 2011 CLSI guidelines

S= Susceptible, I= Intermediate, R= Resistant

Fig. 1- PCR amplification of bla_CTX-M-1 (A), bla_CTX-M-2 (B), bla_CTX-M-8 (C) and bla_CTX-M-9 (D)genes.

 Lanes: L, DNA ladder; 1-8, ESBL E. coli isolates

Table 3- The bla_CTX-M genes in ESBL-producing isolates

+: Producing CTX-M, -: Non-producing CTX-M

CTX-M and antibiotic resistance

For considering the relation between the existence of bla_CTX-M genes and the pattern of antibiotic resistance, the isolates with the higher and lower number of bla_CTX-M genes and also isolates with and without ability to produce CTX-M, were compared according to the profiles of antibiotic resistance (Fig. 2 and Table 4). The two variables didn’t have significant correlation statistically (correlation coefficient or r = 0.245).

The results showed that isolate E1 with one type of CTX-M in contrast to isolates E6, E7 and E8 with three types of CTX-M was sensitive to coamoxyclave, but resistance pattern of isolate E2 was nearly similar to the three isolates mentioned above. Isolate E3 versus isolates E6 and E8, was sensitive to imipenem and chloramphenicol and compared with isolate E7, was sensitive to imipenem only.

Table 4- The resistance pattern of eight ESBL-producing isolates to different antibiotics

E: isolates, R= resistant, I= intermediate and S= sensitive

Fig. 2- The relation between the number of bla_CTX-M genes and the pattern of resistance to antibiotics (number of antibiotics that each isolate was resistant to it)