diarrhea in Khuzestan province, Southwestern Iran
Parviz Owlia
a, Saeid Bouzari
b, Horieh Saderi
a, Roya Ghanavati
cand Atieh Darbandi
d,e,fBackground: DiarrheagenicEscherichia coli(DEC) is an enteric pathogen that causes a wide variety of gastrointestinal diseases in developing countries. In our previous study, the prevalence of DEC pathotypes from acute diarrhea patients in Khuzestan province was determined. In this study, we investigated the antimicrobial resistance profile and molecular genetic characteristics of this isolate.
Methods: Antibiotic susceptibility testing of bacterial isolates was determined by disc diffusion technique on Muller Hinton agar. The production of extended-spectrum beta- lactamases (ESBLs) was confirmed by the Double Disc Synergy Test (DDST). The genetic diversity of isolates was determinate by pulsed-field gel electrophoresis (PFGE).
Results: Among all DEC strains, 100% were resistant to at least one commonly prescribed antibiotic. Strains were resistant to first-line antibiotics, such as tetracycline, ampicillin, and sulfamethoxazole-trimethoprim. Furthermore, 72% of DEC isolates were multidrug-resistant and aEPEC and STEC were the categories with a major proportion of this feature. ESBL-producing strains were observed in 38% of all DEC isolates. PFGE analysis showed 19 unique pulsotypes of 22 studied DEC pathotypes.
However, a few isolates were found to be clonal (clusters A, B, and C).
Conclusion: The current study provides novel information about the presence of DEC isolates particularly with the rate of high antibiotic resistance among acute diarrheal samples in Khuzestan, Iran. Our data revealed that there was almost high heterogeneity among isolated DEC pathotypes. Proper infection control policies are needed to be implemented in order for the infections to be effectively controlled.
Copyrightß2020 Wolters Kluwer Health, Inc. All rights reserved.
Reviews in Medical Microbiology2021,32:106–113
Keywords: antibiogram, diarrheagenic Escherichia coli, extended-spectrum beta-lactamases, pulsed-field gel electrophoresis
Introduction
Infectious acute diarrhea is one of the most important causes of morbidity and mortality in all age groups of people, particularly in children less than 5 years of age [1].
According to recent CDC data, there are approximately four billion cases of diarrhea worldwide. Acute diarrhea
was responsible for around 525 000 deaths of children under 5 years every year [2]. Acute diarrheal diseases are most prevalent in low-income areas in Southeast Asia and Africa, which is responsible for as much as 8.5 and 7.7% of all deaths, respectively [3]. In Iran, infectious diarrhea remains a major public health issue. Microbial agent’s
aMolecular Microbiology Research Center, Shahed University,bMolecular Biology Department, Pasteur Institute of Iran, Tehran,
cBehbahan Faculty of Medical Sciences, Behbahan,dDepartment of Microbiology, School of Medicine, Iran University of Medical Sciences,eMicrobial Biotechnology Research Centre, Iran University of Medical Sciences, andfDepartment of Microbiology, Faculty of Medicine, Shahed University, Tehran, Iran.
Correspondence to Atieh Darbandi, Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
E-mail: [email protected]
Received: 12 April 2020; revised: 26 April 2020; accepted: 27 April 2020
viruses, bacteria, parasites, and fungus can usually cause acute diarrhea [4]. Escherichia coli is one of the most common bacterial agents of infective diarrhea by different functions. The acquiring various functions of E. coli associated with the acquisition of different groups of virulence genes through horizontal gene transfer, resulting in the formation of different pathotypes of diarrheagenic E. coli (DEC). Five pathotypes of DEC included enteropathogenic E. coli (EPEC), Shiga-toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enter- oaggregativeE. coli(EAEC), and diffuse adherentE. coli (DAEC) [5]. EPEC was classified into typical EPEC (tEPEC) and atypical EPEC (aEPEC) according to the presence or absence of EPEC adherence factor plasmid (pEAF), a plasmid that encodes type IV-like bundle- forming pili (BFP) [5]. Diarrhea because of aEPEC is not severely tEPEC as there is no expression pEAF/BFP and lack Shiga toxins encoding genes. ETEC strains cause secretory diarrhea, through the production of the heat-labile (LT) and/or heat-stable (ST) enterotoxins, which is the most common cause of traveler’s diarrhea [6]. EIEC with a mechanism similar to Shigella cause bacillary dysentery in humans by invading epithelial cells of the large intestine and provoking the inflammatory response [5]. EAEC has been associated with persistent diarrhea among patients (adults and children under 12 months) without blood in the feces from industrially developing and developed countries [7]. STEC is associated with bloody diarrhea and hemolytic-uremic syndrome (HUS) through the pro- duction of Shiga toxin, which is a newly emerged pathogen in a group of foodborne strains. The role of DAEC strains in diarrhea remains controversial as the enteric pathogenicity of these strains is still in question [5]. Diarrhea is usually a mild and self-limiting disease and resolves without specific treatment, whereas most cases of acute diarrhea, persistent diarrhea, and acute invasive diarrhea reinforce the use of antimicrobials [8].
Antibiotic treatment plays a crucial role in preventing and treat bacterial infections. Awareness of antimicro- bial resistance of diarrheagenic pathogens is important for effective treatment outcomes. The overuse and misuse of antibiotics in the treatment cause an antibiotic-resistant bacterial infection [9]. In many studies, a different prevalence of DEC strains has been reported from different regions of Iran and other countries [10 – 13]. Despite significant reports of DEC pathotypes from different regions of Iran, there is a paucity of studies revealing the relatedness and/or diversity of the geneticE. colipathotypes. In the study, pulsed-field gel electrophoresis (PFGE) is the bacterial strain typing for genetic fingerprinting, which has slightly higher discriminating power compared with other methods typing [14]. The aim of the present study investigated the antimicrobial resistance profile and molecular genetic characteristics of DEC pathotypes.
Material and methods
Experimental designFecal samples from 200 consecutive acute diarrheal stool specimens were collected from three age groups less than 5, 5 – 14, and more than 14 years and were evaluated for the prevalence of specific DEC for a period of 1 year from March 2012 to February 2013, according to the previous study [15]. The patients were of different backgrounds living in Khuzestan province, Iran [Andimeshk (n¼ 109); Ahwaz (n¼53); and Shooshtar (n¼38)]. Criteria for selection of volunteers included native Iranian patients, lack of any antibiotic therapy over 6 months presampling, not traveling within 2 weeks before the onset of illness, lack of immune deficiency diseases and no use of corticoste- roids and chemical treatment. The diarrheic stool samples were collected aseptically using Cary-Blair (Sigma, UK) transport medium swabs. Prevalence of DEC pathotypes is determined by PCR [15].
Antibiotic susceptibility testing
All DEC isolates were tested for susceptibility to ciprofloxacin (5mg), piperacillin/tazobactam (100mg), ampicillin (10mg), tetracycline (20mg), cefotaxime (30mg), amoxicillin/clavulanic acid (20/10mg), ceftriax- one (30mg), cephalothin (30mg), amikacin (30mg), sulfamethoxazole (10mg), trimethoprim (5mg) and ceftazidime (30mg) by Kirby–Bauer disk diffusion method (MAST, United Kingdom) [16]. Resistance breakpoints to a particular antibiotic using standard reference values. Isolates considered resistance to at least one antibiotic in three or more antibiotic classes were classified as multidrug resistant (MDR) [17]. E. coli ATCC 25922 was included as a quality control organism for antimicrobial susceptibility determination.
Screening and confirmation of extended- spectrum beta-lactamase-producing isolates The presence of extended-spectrum beta-lactamase (ESBLs) was screened phenotypically using cefotaxime (30mg), ceftazidime (30mg), and ceftriaxone (30mg) by standard disk diffusion method on Mueller–Hinton agar.
The presumptive isolates were confirmed by Double Disc Synergy Test (DDST) using cefotaxime (30mg) and ceftazidime (30mg) alone and in combination with clavulanic acid (10mg), as described by the CLSI guidelines [16]. E. colistrain ATCC 25922 was used as negative control and Klebsiella pneumoniae strain ATCC 700603 was used as positive control.
Pulsed-field gel electrophoresis
The DEC isolates that displayed a similar antibiotic resistance profile, were analyzed by pulsed-field gel electrophoresis (PFGE). Genomic DNA patterns of DEC generated by PFGE using XbaI-digested genomic DNA according to the CDC PulseNet protocol [18]. PFGE patterns were then analyzed and interpreted using the Gel
prints were produced by using Dice coefficients and unweighted pair-group method with arithmetic mean (UPGMA), with band position tolerance of 1% and optimization of 1%. XbaI-digestedSalmonella braenderup H9812 DNA was used as the size reference standard.
Statistical analyses
Data were statistically analyzed by employing Pearson chi- square test for dependent samples or Fisher’s exact test whenever appropriate using SPSS software, 19.0 version.
A P-value of less than 0.05 was considered statistically significant.
Ethical considerations
The studies involving human participants were reviewed and approved by Shahed University Ethics Committee.
Results
Identification of Diarrheagenic Escherichia coli pathotypes
According to the previous study, the most frequent pathotype were both EPEC and STEC non-O157:H7 50. The highest prevalence of pathotypes was observed in winter [15].
amongst DEC is presented in Fig. 1. The highest resistance rate was detected for ampicillin 106 (83%), followed by 99 (78%) for trimethoprim/sulfamethoxa- zole and tetracycline; 73 (57%) for cefalotin. On the other hand, most susceptibilities were to amikacin 127 (100%), piperacillin/tazobactam 119 (94%), ciprofloxacin 101 (79%). However, the comparison of resistance patterns between patients belonging to different DEC groups showed that EPEC and STEC strains had a rate of resistance to tested antibiotics. EPEC were significantly more resistant (P<0.05) to ampicillin (90%), tetracycline (84%), Trimethoprim/sulfamethoxazole (78%), cephalo- thin (52%), amoxicillin/clavulanic acid (40%), and ceftazidime (42%). Also, STEC was significantly resistant (P<0.05) to ceftriaxone (46%), cefotaxime (42%), and cephalothin (50%). Of the few EAEC strains isolated, three strains were resistant to tetracycline (60%) but only one to ampicillin (20%).
Multidrug resistance of Diarrheagenic Escherichia coli strains
From all DEC strains isolated, 127 (100%) strains were resistant to at least one antimicrobial agent, 29 different antibiotic resistance patterns are shown in Table 2. Ninet- one (72%) isolates were resistant to three or more antibiotic classes that were identified as multidrug resistance isolates (MDR) with 20 different patterns (X to XXIX) (Table 1). The most prevalent resistance profile
90
80
57
20
0 40
26
9
0 0
42 38
0 0 0
36
46
0 0 0
10 4 0 0 0
34
42
0 0 0
52 50
0 0 0
20 22
0 0 0
0 0 0 0 0
84
66
9
60
0 78
62
33
0 0
0 10 20 30 40 50 60 70 80 90 100
EPEC STEC ETEC ETEC ETEC
Percent (%)
Diarrhea E. coli pathotypes Resistance antibiotics profile
Ampicillin Amoxicillin /Cluvlanic acid Ceftazidime
Ceftriaxone Piperacillin/tazobactam Cefoxitin
Cephalothin Ciprofloxacin Amikacin
Tetracycline Trimethoprim/sulfamethoxazole
Fig. 1. Antimicrobial resistance among diarrheagenicEscherichia coliisolates.
of MDR was XIII or XXVII (8%) (ampicillin, amoxicillin /clavulanic acid, cephalothin, tetracycline, trimetho- prim-sulfamethoxazole and ampicillin, amoxicillin/cla- vulanic acid, ceftazidime, ceftriaxone, ceftazidime, cephalothin, ciprofloxacin, tetracycline, trimethoprim- sulfamethoxazole, and ampicillin). Among the acute diarrheagenic isolates of MDR, 31 (24%) ofE. coliwere resistant to five antibiotics followed by 24 (19%) strains
were resistant to 7 antibiotics, 10 (8%) to 7, 10 (8%) to 9, and 9 (7%) to 3 antimicrobial agents, 4 (3%) to 4 antibiotics, 2 (2%) to 8 antibiotics, and 1 (1%) to 10 antibiotics had less frequent resistance phenotype (Table 2). The prevalence of MDR phenotype among DEC pathotype demonstrate that EPEC (88%) or STEC (70%) strains were significantly higher than ETEC (62%) (P<0.05). Two (40%) of EAEC strains showed a
Table 1. Distribution of resistance patterns among diarrheagenicEscherichia colistrains isolated from patient with acute diarrhea in Khuzestan, Iran, March 2012 to February 2013.
DiarrheaEscherichia colipathotypes Age groups (years)
Antibiotics Resistance pattern EPEC STEC ETEC EAEC EIEC Total <5 5—14 >14
AMP I 0 3 3 0 0 6 1 2 3
SXT II 0 0 2 0 0 2 0 0 2
TE III 0 0 2 1 0 3 0 0 1
TE, SXT IV 3 5 0 0 1 9 3 2 4
TE, CF V 3 0 0 0 0 3 1 0 2
AMP, CTR VI 0 4 0 0 0 4 0 0 4
AMP, TE VII 0 3 0 0 0 3 1 1 1
AMP, SXT VIII 0 0 2 1 0 3 2 0 2
AMP, CAZ IX 0 0 2 1 0 3 0 0 3
AMP, TE, SXT X 3 3 0 0 0 6 2 0 4
AMP, AMC, SXT XI 0 0 3 0 0 3 0 0 3
AMP, CAZ, TE, SXT XII 4 0 0 0 0 4 1 1 2
AMP, AMC, CF, TE, SXT XIII 3 5 2 0 0 10 4 1 5
AMP, CAZ, CF, TE, SXT XIV 3 0 1 0 0 4 0 1 0
AMP, CTR, CF, TE, SXT XV 3 3 1 0 0 7 2 2 4
AMP, AMC, PTZ, CTX, CP XVI 0 2 2 1 0 5 2 1 2
AMP, CAZ, CTR, CTX, CF XVII 0 3 1 1 0 5 2 1 2
AMP, CAZ, CTR, CTX, CF, SXT XVIII 0 3 0 0 0 3 0 1 2
AMP, AMC, CAZ, CTX, TE, SXT XIX 0 2 0 0 0 2 1 1 0
AMP, AMC, CTX, CF, TE, SXT XX 3 0 0 0 0 3 1 1 1
AMP, AMC, CAZ, CF, TE, SXT XXI 3 0 0 0 0 3 1 1 2
AMP, CTR, CTX, CF, TE, SXT XXII 5 2 0 0 0 7 2 2 3
AMP, CAZ, CTR, CF, TE, SXT XXIII 4 2 0 0 0 6 2 2 2
AMP, AMC, CAZ, PTZ, CF, TE, SXT XXIV 3 0 0 0 0 3 0 0 3
AMP, AMC, CTX, CF, CP, TE, SXT XXV 3 0 0 0 0 3 1 0 2
AMP, CAZ, CTR, CTX, CF, TE, SXT XXVI 2 2 0 0 0 4 1 4 2
AMP, CAZ, CTR, CTX, CF, CP, TE, SXT XXVII 0 2 0 0 0 2 1 1 1
AMP, AMC, CAZ, CTR, CTX, CF, CP, TE, SXT
XXVIII 4 6 0 0 0 10 2 2 4
AMP, AMC, CAZ, CTR, PTZ, CTX, CF, CP, TE, SXT
XXIX 1 0 0 0 0 1 0 0 1
Total 50 50 21 5 1 127 33 27 67
AK, amikacin; AMC, amoxicillin/cluvlanic acid; AMP, ampicillin; CAZ, seftazidime; CF, cephalothin; CIP, ciprofloxacin; CRO, ceftriaxone; CTX, cefotaxim; PTZ, piperacillin-tazobactam; SXT, trimethoprim-sulfamethoxazole; TE, tetracycline.
Table 2. Multiple antimicrobial resistance of diarrheagenicEscherichia colistrains isolated from acute diarrhea cases.
DEC strain [n(%)]
Resistant tonantibiotics Total 127 EPEC 50 (39) STEC 50 (39) ETEC 21 (16) EAEC 5 (4) EIEC 1 (1)
0 0 0 0 0 0 0
1 11 (9) 0 3 7 1 0
2 25 (20) 6 12 4 2 1
3 9 (7) 3 3 3 0 0
4 4 (3) 4 0 0 0 0
5 31 (24) 9 13 7 2 0
6 24 (19) 15 9 0 0 0
7 10 (8) 8 2 0 0 0
8 2 (2) 0 2 0 0 0
9 10 (8) 4 6 0 0 0
10 1 (1) 1 0 0 0 0
DEC, diarrheagenicEscherichia coli.
were no significant statistical differences between three age groups of the patients and rates of antibiotic resistance (P¼0.312). There were no significant differences for incidence of antibiotic resistance between the male and female individuals (P¼0.432).
Determination producing strains of extended- spectrum beta-lactamases
Among 56 isolates (44%) that were suspected as ESBL- producers; only 48 (38%) of the isolates were confirmed as producing ESBL by double-disc synergy test. Among pathotypes, 24 (50%) STEC (most prevalent among >5 years of age), 21 (44%) EPEC (most prevalent among<5 years of age), and 3 (6%) ETEC (most prevalent among>14 years of age) were ESBL producer (Fig. 2). ESBL producers was not detected in EAEC pathotypes. All ESBL-producing isolates were also MDR. The resistance rates of ESBL- producing strains toward third-generation cephalosporins, such as ceftazidime (96%), followed by cephalothin (90%), ceftriaxone (79%), and cefotaxime (63%), were significantly higher than in non-ESBL-producing strains (P<0.05) (Fig. 3). Our findings also showed that resistance of ESBL isolates was resistant to none–b-lactam antibiotics, for example, ampicillin (96%), trimethoprim-sulfamethoxazole (90%), tetracycline (79%), and ciprofloxacin (37%) (Fig. 3).
The high percentage of ESBL-positive isolates was found to be susceptible to amikacin (100%), piperacillin/tazobactam (10%) (Fig. 3). The results of antimicrobial susceptibility testing of the ESBL-producing DEC isolates are summa- rized in Fig. 2. There were no significant statistical differences between the age or sex of the patients and the rates of ESBL producers (P¼0.613).
Pulsed-field gel electrophoresis
The 22 DEC isolates (nine EPEC, nine STEC, three ETEC, and one EAEC) that showed similar resistance antibiotics profile were analyzed by PFGE to determine
their genetic relationships. High heterogeneity was demonstrated within isolates of DEC. At a genetic similarity of 80%, three clusters were formed, designated as A_C with two isolates per cluster. Also, 19 unique PFGE profiles were obtained from the genome of pathotypes (cluster 1 to cluster 19). They were named from G1 to G19. Of these three clusters, EPEC isolates from cluster B (G19 and G20) were also exhibiting 80%
genetic similarity (Fig. 4). Interestingly, EPEC and STEC isolates of cluster A (G7 and G17) and EPEC and EAEC of cluster C (G21 and G22) were also exhibiting 80%
genetic similarity. Also, three clusters were exhibiting almost similar antibiotic resistance profiles for tested antimicrobial agents and were showing more than 70%
resistance similarity (Table 3).
Discussion
Among bacterial agents, diarrheagenic E. coli (DEC) is one of the most common etiological causes of acute infectious diarrhea among adults and children worldwide by different mechanisms. Each different pathotype ofE.
coli had different virulence factors and pathogenic mechanisms associated with human disease. The preva- lence of different pathotypes of DEC is well documented in a regional difference in Iran [13,19,20]. In this regard, an epidemiological knowledge of the disease-causing agent is essential in order to better handle disease outbreaks and therapeutic strategies. Here, we studied genetic diversity and the antimicrobial sensitivity profile of DEC pathotypes collected from the acute diarrhoeic humans. In our previous study, aEPEC and STEC identified as the most common pathotype among acute
0 2 4 6 8 10 12 14 16
< 5 5 - 14 > 14
Number of ESBL producer
Age groups (years) EPEC STEC ETEC
Fig. 2. The prevalence of extended-spectrum beta-lacta- mase-producing isolates among different age groups. No significant relationship was found between extended-spec- trum beta-lactamase (ESBL)-producing isolates and different age groups (P>0.05).
0 20 40 60 80 100
AMP CAZ CRO CTX CF AMC PTZ CIP AK TE SXT
Percent (%)
Antibiotic Sensitive Resistance
Fig. 3. Antibiotic resistance profiles among isolates of extended-spectrum beta-lactamase producer.AK, amikacin;
AMC, amoxicillin/cluvlanic acid; AMP, ampicillin; CAZ, ceftazidime; CF, cephalothin; CIP, ciprofloxacin; CRO, cef- triaxone; CTX, cefotaxim; PTZ, piperacillin-tazobactam; SXT, trimethoprim-sulfamethoxazole; TE, tetracycline.
diarrhea samples of different age groups from Khuzestan province, Iran [15]. Other studies have reported various prevalence rates of DEC pathotypes [12,13,19–21].
Apart from infection with a single pathotype, mixed co infections showed were observed in both children (n¼3)
and adults (n¼1). Various mixture of DEC co-infection in diarrheal samples was also reported in other studies [13,22,23]. This may be because of the influence of different regions in the distribution of DEC and within countries in the same region as well as the time of
Table 3. Pulsed-field gel electrophoresis and antibiogram profile of diarrheagenicEscherichia colipathotypes isolate.
Antibiogram of the DEC pathotypes in clusters
ID Phatotype PFGE patterns City Sex Age AMP CRO CF CTX CAZ AMC PTZ CIP AK TE SXT ESBL
17 EPEC G1 Andimeshk Female 53 R R R R R R S S S R R -
7 STEC G2 Ahvaz Male 2 R R R R R R S S S R R -
11 EPEC G3 Ahvaz Male 2 R S S S S S S S S R R þ
16 ETEC G4 Ahvaz Male 32 R S S S S S S S S S S þ
10 ETEC G5 Ahvaz Male 6 R S S S S S S S S S R -
12 ETEC G6 Ahvaz Male 62 R S S S R S S S S S S þ
6 STEC G7 Andimeshk Female 23 R S S S S S S S S S S þ
13 EPEC G8 Andimeshk Male 1.5 R S R S S R S S S R R -
9 STEC G9 Andimeshk Male 8 S S S S S S S S S R R þ
1 STEC G10 Ahvaz Male 43 R S S S S S S S S R R -
15 EPEC G11 Andimeshk Female 19 R S S S R S S S S R R -
18 EPEC G12 Ahvaz Male 3 R R R S S S S S S R R þ
19 EPEC G13 Ahvaz Male 2 R R R S R S S S S R R -
20 EPEC G14 Andimeshk Female 53 S R R R R R S S S R R -
14 EPEC G 15 Ahvaz Male 6 R R R R R S S S S R R þ
8 STEC G16 Andimeshk Male 16 R S S S S R S S S R R -
21 EPEC G17 Andimeshk Male 34 S S S S S S S S S R R þ
22 EAEC G18 Ahvaz Male 3 S S S S S S S S S R S þ
5 STEC G19 Andimeshk Male 17 R R R S S S S S S R R -
2 STEC G20 Andimeshk Male 47 R S S R S R R R S S S -
4 STEC G21 Ahvaz Female 2 R R R R R S S S S S R -
3 STEC G22 Shoshtar Male 3 R S S S S S S S S R S -
AK, amikacin; AMC, amoxicillin/cluvlanic acid; AMP, ampicillin; CAZ, ceftazidime; CF, cephalothin; CIP, ciprofloxacin; CRO, ceftriaxone; CTX, cefotaxim; DEC, diarrheagenicEscherichia coli; PTZ, piperacillin-tazobactam; R, resistant; S, sensitive; SXT, trimethoprim-sulfamethoxazole; TE, tetracycline.
Fig. 4. Pulsed-field gel electrophoresis profile of diarrheagenicEscherichia colipathotypes isolated from acute diarrhea.The genotypic patterns generated by pulsed-field gel electrophoresis were analyzed the gel compare II, version 6.5 software (Applied Maths, Belgium). The clustering was performed by UPGMA and the dice correlation coefficient.
showed a high rate of antimicrobial resistance. In recent decades, overuse and misuse have resulted in an increased prevalence of resistance to these antibiotics. The resistance rates of DEC to traditional antibiotics were high, for example, such as ampicillin (77%), and tetracycline (62%), and trimethoprim/sulfamethoxazole (61%). Our findings are in agreement with other studies showed universal low activity of these three agents against the DEC strains in Iran and other countries [11,24]. Furthermore, in developing countries, such as Iran, these antibiotics are used to treat diarrhea because of their low cost and availability. We suggest that these three antibiotics should not be used as a first-line therapeutic drug for Enterobacteriaceae. Despite the high prevalence of antimicrobial resistance observed among DEC isolates, these organisms remain highly susceptible to amikacin antibiotics (100%), piperacillin/
tazobactam (95%), ciprofloxacin (84%), which are now the drugs of choice in many areas. Similar to this study, Borujerdiet al.from Khuzestan reported high sensitivity (>70%) of DEC isolates to imipenem, ciprofloxacin, and amikacin [13]. The prevalence of MDR DEC in this study was 72%. In recent years, the high incidence rate of MDR- DEC isolates reported in developing countries including China, and India [12,25].
Also, the high rate of multidrug antibiotic resistance showed among DEC isolates to the antibiotics that most frequently used to treat diarrheal illnesses, such as ampicillin, tetracycline, and TMSX in Iran. This trend has been observed in the study of Alikhanet al.[11]that reported 67%
of MDR DEC strains isolated from adults were resistant for ampicillin, co-trimoxazole, and tetracycline. Also, other studies reported prevalence of resistance for ampicillin, co- trimoxazole, and tetracycline among MDR -DEC isolated from the children population [10,11,24,26]. The most prevalent multidrug resistance patterns demonstrated emergency of isolates resistant to more than five antibiotics.
These findings illustrate the need for monitoring and surveillance systems for MDR in Iran. The prevalence of resistance to the drugs could be explained by the fact that this drugs has been used to treat disease infection for a long time, therefore, ensuring selection pressure for the maintenance of resistance. So, continuous surveillance of antibiotic resistance pattern is needed for the development and implementation of best practices strategies for antibiotic usage. The resistance rate of DEC to cephalosporins especially third-generation cephalosporin is concerning, and should not be ignored, because of the presence of isolates producing beta-lactamase. ESBLs are capable of hydrolyzing third-generation cephalosporins, but not carbapenems, and therefore can be inactivated by clavulanic acid. The prevalence of ESBL production among DEC isolates was lower in this study than in some previous Iranian studies [13] and higher than other reports [21,27].
The differences in the prevalence of ESBL might be because of the difference in sample size, dissimilarities in
were significantly higher than in non-ESBL-producing strains. This is worrying, since that ESBL-producing strains are often associated with mobile genetic elements, which can harbor genes encoding for resistance to antibiotics [28].
The molecular pattern of DEC showed a high degree of polymorphic, indicating that the human cases were most likely sporadic, as found in another study. In this study, based on Tenover and his colleague’s guidelines, we observed three clusters designated as A–C, and of these three clusters, one cluster was represented by EPEC isolates (cluster B) recovered from patients with different age groups of 2 years and 53 years in two cities of Khuzestan province. This indicates that a single strain with a similar resistance profile was circulating in these two cities of Khuzestan province.
Under this circumstance, effective infection control programs should be more regarded; otherwise, the dissemination of pathogenic strains to another city would be expected. The same status was also observed in two clusters (A and C), but with different pathotype (EPEC and STEC, EPEC and EAEC, respectively). This result indicates that the two clusters belonging to different pathotypes are closely related. Circulation of closely related strains with different pathogenicities among patients with different ages from two cities contributes to the fact that they could disseminate in every setting leading to critical problems for public health. This finding also supports that the clustered DEC isolates closely related to each other;
however, further studies with more numbers of isolates of the different sources may provide a useful marker for future epidemiological and intervention studies.
In conclusion, the current study provides novel informa- tion about the presence of DEC isolates particularly with the rate of high antibiotic resistance among acute diarrhea samples in Khuzestan, Iran. Our data revealed that there was almost high heterogeneity among isolated DEC pathotypes. however, a few DEC pathotypes were found to be clonal, suggesting that these DEC pathotypes might have been circulated between human and associated environmental sources. In addition to this, all DEC pathotypes not only exhibited a high degree of antimicro- bial resistance profile but also showed a high frequency of ESBL. These properties enable DEC can resist the effects of medication that once could successfully treat the DEC.
Furthermore, it suggests that clinical misuse of antibiotics is a very serious issue in Iran. However, further investigations are needed including epidemiological triad as a potential area for future research work.
Acknowledgements
Conflicts of interestThere are no conflicts of interest.
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