Detection of blaTEM, blaSHV, and blaCTX-M genes among the Extended-Spectrum β-Lactamases (ESβLs) producing Enterobacteriaceae isolated from hospital-acquired infections and community in Egypt.

INTRODUCTION

Bacteria are resistant to an antibiotic such as Escherichia coli, Klebsiella pneumonia, and Enterobacter sp. responsible for morbidity in worldwide¹. Antibiotics resistance due to extended-spectrum β-lactamases (ESβLs) was firstly recorded in 1979 in Europe². ESβLs are a group of plasmids encoded enzymes that have the efficacy to hydrolyze β-lactams antibiotics³. It was firstly reported that ESβLs producers are predominantly belonging to E. coli and Klebsiella ⁴

Among several enzymes’ linkage with ESβLs activity, ESβLs class A include on cefotaximase (CTX-M), Temoneira  (TEM), and SHV (to sulfhydryl variable active site)^3. These genes are commonly in Klebsiella pneumonia and E. coli ⁵. Investigation of these genes is important not only for their ability to hydrolyze β-lactam antibiotics but also because the plasmids responsible for ESβLs production regularly harboring genes encoding resistance to other antibiotic groups like aminoglycosides and fluoroquinolones. A previously published study by Hassan et al.,2012 reported 98% of 65 Klebsiella pneumonia isolates obtained from Egyptian patient samples harbor SHV gene while 11% harbor CTX-M gene ⁶. In another study conducted in Egypt highlighted that the CTX-M gene is the predominant resistance gene in ESβLs Enterobacteriaceae ⁷. To date, few published studies were concerned with the assessment of ESβLs resistant genes in Enterobacteriaceae strains of the hospital and community setting. Therefore, this study aimed to isolate Enterobacteriaceae producing ESβlsspecies from rectal swab samples, antibacterial resistance pattern and determine the predominate ESβLs resistance gene in isolates of hospital settings versus the presence of those genes in rectal isolates from community settings

PATIENTS AND METHODS

Samples collection

One hundred rectal swab samples were collected from 50 patients (two samples taken from each patient, one upon admission and second after 48h of admission) at Abu El-Reesh Pediatric Hospital, Cairo University Hospital, Egypt, and one hundred rectal swab samples from healthy individuals duration period extending from December 2016 to December 2019.

Cultivation and isolation of bacterial species

Rectal swab samples were cultivated on MacConkey media and incubated for 24 hours at 37^oC aerobically. Colonies with positive lactose fermentation (Pink colonies) were collected. The pure cultures were identified based on morphological, physiological, and biochemical characteristics using microbiological methods 8^th, ⁸ Bergey’s Manual of Systematic Bacteriology ⁹. Isolates identification was confirmed by the VITEK2 compact system (Biomerieux Inc., Marcy I’Etoile, France).

Screening of (ESβLs) production

 The antibiotics synergy of ESβLs producing bacteria was detected by the double-disk synergy test (DDST) ¹⁰.Bacterialcolonies from MacConkey agar equivalent to 0.5 McFarland are cultured on Mueller-Hinton agar media. The following antibiotic discs are used, cefotaxime 30μg/ml, ceftazidime 30μg/ml (third-generation cephalosporins), and amoxicillin/clavulanate 20/10μg/ml. Culturing plates were incubated at 35^oC for 24h. ESβLs production activity is confirmed if there is an extension of the inhibition zone between any of the cephalosporins and amoxicillin-clavulanate disk (D-shape or keyhole shape) ^{11, 12}.

Antibiotic resistance pattern

Antibacterial sensitivity testing was carried out by disc diffusion method and the results were expressed as resistant, intermediate, or susceptible according to CLSI guidelines ¹⁰. The antibiotics used in this study belonging to four groups antibiotics carbapenems include on (imipenem10μg and meropenem10μg), aminoglycosides (gentamicin 10μg/ml andamikacin 30μg/ml), fluoroquinolones (ciprofloxacin 5μg/ml and levofloxacin 5μg/ml), and polypeptides (colistin10μg/ml and polymyxin B 300U/ml).

Molecular detection of bla_TEM, bla_SHV, and bla_CTX-M genes using PCR.

A total of 120  ESβLs producing Enterobacteriaceae were investigated for detecting three genes (bla_TEM, bla_SHV, and bla_CTX-M) using multiplex PCR and specific primers (Table 1). The detection methods were designed according to the methods Randall et al., 2009 ¹³.

Table 1:. Primer sets used in PCR runs for tested isolates.

Statistical analysis:

The data were subjected to analysis of variance (ANOVA) by statistical package SPSS v17. The mean difference comparison between the treatments was analyzed by the Tukey HSD test at a significance level of P ≤ 0.05.

RESULTS

Screening of ESβLs producing Enterobacteriaceae

A total of 200 Enterobacteriaceae were screening for producing ESβLs, the results obtained showed that 112 (56%) bacterial isolates producing ESβLs. Thirty-two isolates (64%) from patients upon admission, 40 (80%) from the same patients after 48h of admission, and 40 (40%) from healthy individuals.

identification of ESBLs Enterobacteriaceae

The results revealed that 112 bacterial isolates producing ESβLs (32 isolates from patients upon admission, 40 from same patients after 48h of admission, and 40 from healthy individuals) were included to identify. The results obtained from morphological, physiological, and biochemical tests revealed that 100% of bacterial isolates are Gram-negative, rod shape, motile, ferment lactose sugar in MacConkey agar, and positive results in the triple sugar iron (TSI) test.  Only 46.4% (52/112) from bacterial isolates have the ability to utilizing tryptophan and forming indole and produce decarboxylase enzyme, Moreover, 1.8% (2/112), 51.73% (58/112), and 53.5(60/112) can produce H₂S, urease, and citrate utilization respectively (Table 2). According to results obtained from the identification of 112 ESβLs - producing Enterobacteriaceae species, the most common species was found Klebsiella pneumonia followed by E. coli and Enterobacter cloacae with percent 51.8% (58/112),  46.4% (52/112), and 1.8% (2/112), respectively.  This result was confirmed by VITEK-2 with an echelon ratio of 99%. In this study, The identification of bacterial species isolated from healthy individuals revealed that the most common species E. coli 22.3% (25/112) followed by Klebsiella pneumonia 13.38% (15/112), comparable isolates from patients the most common species were found Klebsiella pneumonia 38.35% (43/112) followed by E. coli 24% (27/112) and 1.8% (2/112) Enterobacter cloacae.

Table 2: Identifications of 112 ESβLs producing Enterobacteriaceae species isolated from patients and healthy individuals.

Resistance patterns of  Enterobacteriaceae

The antibiotic profile of Enterobacteriaceae isolated from patients showed the highest resistance to ciprofloxacin, levofloxacin, and amikacin (71.76%), (60.72%) and (60.72%), while, they were sensitive to colistin, polymyxin, meropenem, and imipenem (95.22%), (95.22%) (44.16%), and (44.16%), respectively. Moreover, it was noted that bacterial species isolated from patients after 48h of admission highly resistant to bacterial species isolates from the same patients upon admission and healthy individuals (Table 3). E. coli isolated from patients after 48hours of admission showed the highest resistance to ciprofloxacin, levofloxacin 91.63%, while, isolates from patients upon admission 66.6%. However, E. coli isolated from healthy individuals exhibit a low resistance level  (Table 4). In K. pneumoniae, from patients after 48hours of admission the highest resistance was observed against and ciprofloxacin, levofloxacin 81.4% with a low resistance level to polymyxin B  and colistin 3.70%. (Table 5). Enterobacter cloacae recorded the highest resistance to ciprofloxacin, levofloxacin with 100%. K. pneumoniae showed the resistance to meropenem and imipenem with 53.32%, 51.6%, followed by Enterobacter cloacae 50.0% and E. coli with 23.04%, respectively (table 4 and 5). Overall, K. pneumoniae showed the highest resistance level from E. coli isolates (Table 6).

Prevalence of bla_TEM, bla_SHV, and bla_CTX-M genes in Enterobacteriaceae species

In our study,  88/112 (78.5%)  isolates it has bla_CTX-M resistant gene, 82/112(73.2%)  bla_TEM gene and 77/112 (68.70%)  bla_SHV  (table 7) Figure 1A, 1B, 2A and 2B.

The statistical analysis revealed that no significant statistical difference between the presence resistant genes bla_TEM, bla_SHV, and bla_CTX-M in healthy individuals isolates comparable with isolates collected from patients either upon admission or after 48h. of admissions, Also, the difference between the presences of the three genes in bacterial isolates collected from patients cases either upon admission or after 48h of admission was statistically insignificant (Table 8). Interestingly, the predominant resistant gene in bacterial species isolated from patients and healthy individuals is the bla_CTX-M gene followed by bla_TEM and bla_SHV (Table 8). bla_CTX-M was the predominant gene in Klebsiella pneumonia isolates (81.0%) while it was the second common in E. coli isolates (75.0%). The bla_CTX-M resistance gene was the predominant gene in E. coli isolates (80.8%), and the least gene in Klebsiella pneumonia isolates (67.2%), bla_SHV, a second common gene in Klebsiella pneumonia (79.3%) (Table 9).

Table 3: Antibiotic resistance pattern of 112 ESβLs producing Enterobacteriaceae species.

Table 4: Antibiotics resistance pattern of E. coli    R= Resistant, I= Intermediate, S= Sensitive.

Table 5: Antibiotics resistance pattern of Klebsiella pneumonia   R= Resistant, I= Intermediate, S= Sensitive.

Table 6: Antibiotics resistance pattern oftotal E. col, Klebsiella pneumonia, and Enterobacter cloacae. R= Resistant, I= Intermediate, S= Sensitive

Table 7: Summary of prevalence bla_CTX-M, bla_TEM, bla_SHV genes in the 112 ESβLs producing Enterobacteriaceae species.

Table 8: Distribution of bla_CTX-M, bla_TEM, bla_SHV genes in ESβLs producing Enterobacteriaceae species.

Table 9: Distribution of bla_CTX-M, bla_TEM, bla_SHV genes in ESβLs-producing Enterobacteriaceae.