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Antimicrobial resistance patterns of diarrheagenic and nondiarrheagenic Escherichia coli isolates from Libyan children Abstract

2014 Vol. 4 No. 1:2 doi: 10.3823/744

Entisar Alnagi Omran1,2, Azmi M. Mahafzah2, Asem A. Shehabi2 1 Benghazi Children Hospital, Libya and 2 Department of Pathology-Microbiology Faculty of medicine, The Jordan University, Amman, Jordan.

Corresponding author:

Background: Diarrhea continues to be one of the most common cause of morbidity and mortality among infants and children in developing countries. This study was carried out among Libyan children hospitalized in Benghazi children hospital, to be investigated for the incidence of diarrheagenic E.coli types and antimicrobial susceptibility profiles of all E.coli isolates in their stool samples.

Prof. Asem A. Shehabi

 [email protected]  [email protected]

Methods and patients: A total of 200 children with diarrhea and 90 without diarrhea were their stools investigated using culture on MacConkey agar, and the E.coli isolates were examined for detection of diarrheagenic E. coli types, antimicrobial susceptibility pattern, presence of integrons, ESBL-CTX-M and fluroquinolone resistance genes using PCR. Results: The study has indicated that diarrheagenic E. coli isolates were found only in stools of children with diarrhea. The detected diarrheagenic E. coli types were the following: 4 (2%) enteroaggregative E. coli (EAEC), 3 (1.5%) enteroinvasive E. coli (EIEC), and 1(0.5%) enterohemorrhagic E. coli (EHEC), and both enterotoxigenic E. coli(ETEC) and enteropathogenic E. coli(EPEC) were not present. It has also found that multidrug resistant E.coli (> 3 drugs) was higher prevalent in commensal intestinal E.coli than in diarrheagenic E.coli isolates, and both carried high rates of Class 1 integrons, bla(CTX-M) and fluroquinolone resistance genes. Conclusion: This study revealed the low incidence of diarrheagenic E. coli isolates and high prevalent of antimicrobial resistance among normal intestinal E.coli of hospitalized children in Benghazi/Libya. Key words: Diarrheagenic E.coli, antimicrobial-resistance genes, Libya. © Under License of Creative Commons Attribution 3.0 License

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Introduction

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and which has a high sensitivity and specificity for their detection [12,13].

Infectious diarrhea continues to be one of the most common cause of morbidity and mortality affecting children in many developing countries. It is the second leading cause of death in children under five years old, and both Rotavirus and diarrheagenic E.coli are the leading cause of diarrheal disease [1]. Among the bacterial pathogens, diarrheagenic Escherichia coli (E.coli) is an important agent of endemic and epidemic diarrhea worldwide and especially in developing countries [1,2]. Among the E. coli causing diarrhea, there were five commonly recognized types : enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), enterohemorhagic E. coli (EHEC). Each type has specific virulence factors that are used for their identification and classification. With the exception of EHEC , especially E. coli 0157:H7 , diarrheagenic E. coli strains are not routinely detected in most clinical laboratories [2]. Diarrheal disease caused by E.coli are usually transmitted through food or water contaminated with human or animal feces [3,4]. Person-to-person transmission might also take place, but it is probably less common. There are few studies from the Arab countries including Libya which have investigated the occurrence of diarrheagenic E.coli genotypes and their antimicrobial susceptibility profiles during the last 10-year [5-11]. Accurate detection and identification of diarrheagenic E.coli can’t be done only by culture, biochemical and serotyping tests, since they are indistinguishable from the non- pathogenic E.coli commonly found in human feces. Therefore, only DNA based method such as polymerase chain reaction (PCR) assay can be used for rapid and reliable diagnosis,

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2014

The present study carried out with the aim to determine the incidence of diarrhoeagenic E. coli in hospitalized children in Benghazi Children Hospital, Libya, using multiplex PCR , and to investigate the antimicrobial resistance profiles of commensal intestinal E.coli isolates in these children.

Patients & Methods Patients A total of 290 stool samples were collected from hospitalized children in the Children Hospital of Benghazi, Libya, over a period of 3 months (March to June 2012). Of these, 200 stool samples were obtained from patient with diarrhea and 90 samples from healthy controls without diarrhea. The study was approved by the administration of Benghazi Children Hospital, Libya. An informed consent was obtained from parents. Data collection included the following: age, gender, presence or absence of watery or bloody diarrhea, fever, antibiotic treatment, and presence of specific associated disease.

Collection of stool samples Stool samples were collected from children using a cotton swabs and these were directly inoculated into transport fluid medium. All samples were transferred to the microbiology laboratory for culture in the laboratory of Benghazi Children Hospital.

Isolation and Confirmation the identity of E.coli isolates Each fresh stool sample was cultured directly on MacConkey agar plate (Oxoid, Basingstoke, Eng-

Received 10 June 2014; accepted 6 July 2014



land) and incubated aerobically at 37c’ for 18-24h. A total of 10 red colonies that morphologically suspected to be E.coli were picked up and incubated in 1 ml brain-heart infusion broth tubes which contained 25% glycerol, and the tubes were stored at -70Cº for further investigation. All other investigations were carried out at the Research Microbiology Laboratories, Faculty of Medicine, The Jordan University, Amman, Jordan. All collected E.coli isolates were identified first on basis of lactose fermentation on MacConkey, biochemical tests including fermentation of Triple sugar iron agar test(TSI), positive indole and citrate utilization test, and negative urease production.

2014 Vol. 4 No. 1:2 doi: 10.3823/744

Multiplex PCR reaction for detection Diarrheagenic E. coli DNA extraction was performed using G-spin Total DNA Extraction Kit ( INTRON Biotechnology / Gyeonggi-do 462-807 Korea). The DNA extracts of investigated E.coli isolates were used after spectrophotometric quantitation for detection of the following markers: eaeA (structural gene for intimin of EHEC and EPEC), bfpA (structural gene for EPEC), vt1 and/or vt2 (Shiga toxins 1 and 2 of EHEC), eltB and/or estA (enterotoxins of ETEC), ial (EIEC), and pCVD (EAEC) as described by Shehabi et al. [11]. The primers selected for use in the amplification are matched the sequences of the corresponding genes of EAEC, EHEC, EIEC, EPEC, and ETEC in the GenBank and EMBL database libraries as shown in Table 1.

Table 1. Primers used for detection of diarrheagenic E.coli and antimicrobial resistant genes. Primer

Target gene

LT

eltB

ST

estA

VT1

vt1

VT2

vt2

Eae

eaeA

SHIG

Ial

bfpA

bfpA

EA

PCVD

Primer sequence (5’-3’) 5-TCTCTATGTGCATACGGAGC-3 5-CCATACTGATTGCCGCAAT-3 5-GCTAAACCAGTAG AGGTCTTCAAAA-3 5-CCCGGTACAG AGCAGGATTACAACA-3 5-GAAGAGTCCGTGGGATTACG-3 5-GAAGAGTCCGTGGGATTACG-3 5-ACCGTTTTTCAGATTTTGACACATA-3 5-TACACAGGAGCAGTTTCAGACAGT-3 5-CACACGAATAAACTGACTAAAATG-3 5-AAAAACGCTGACCCGCACCTAAAT-3 5-CTGGTAGGTATGGTGAGG-3 5-CCAGGCCAACAATTATTTCC-3 5-TTCTTGGTGCTTGCGTGTCTTTT-3 5-TTTTGTTTGTTGTATCTTTGTAA-3 5-CTGGCGAAAGACTGTATCAT-3 5-CAATGTATAGAAATCCGCTGTT-3

© Under License of Creative Commons Attribution 3.0 License

Amplicon size (bp) 322 147 130 298 376 320 367 630

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Antimicrobial Susceptibility testing

Results

Antimicrobial susceptibility testing of E.coli isolates was performed by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar medium (Oxoid, Basingstoke, England), MICs of 4 antimicrobials were determined using Etest® (BioMe´rieux Inc., France). The results were interpreted according to the recommendation guidelines of the Clinical Laboratory and Standards Institute (CLSI,2012) (14 ). In addition, the isolates were investigated for presence of integrons (classes I, II, and III) and antibiotic-resistance genes of bla(CTX-M) as reported by AbSalah et al. 2013 [15]. The antimicrobial drugs tested are shown in Table 3. E. coli ATCC 25922 strain was included as a control in all laboratory assays.

This prospective study investigated a total of 290 hospitalized children with and without diarrhea (control group) aged between one month and approximately 48 months. The 200 patients with diarrhea included 115 (57.5%) males and 85 (42.5%) females divided according their age in 7 groups as shown in Table 2. A total of 62% of children with watery / bloody diarrhea and have fever were aged ≤ one year. The control group included 90 children without diarrhea, of these 50 (55.6%) were females and 40 (44.4%) were males (Table 2).

Table 2. Distribution of age among the two groups of examined children.

Total 200 90 290

No. (%) Age/months 37-48

31- 36

25 -30

19 -24

13- 18

7-12

1-6

14 (7) 4 (4.4)

8 (4) 7 (7.8)

7 (3.5) 12 (13.3)

15 (7.5) 16 (17.8)

32 (16) 19 (21.1)

77 (38.5) 16 (17.8)

47 (23.5) 16 (17.8)

18

15

19

31

51

93

63

Children group Diarrhea Control Total

Table 3. Distribution of diarrheagenic E.coli types in stools of 290 children. No. (%) Children

Diarrheagenic E. coli

Without diarrhea**

With diarrhea*

0

3 (1.5)

EIEC

0

1 (0.5)

EHEC

0

4 (2)

EAEC

0

0

EPEC

0

8 (4)

Total

* 102 (51%) and ** 41(45.6% ) were on antibiotic treatment during collection of stools

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Vol. 4 No. 1:2 doi: 10.3823/744

The following diarheagenic E.coli isolates were recovered from all examined 290 stool samples of children; 4 (2%) enteroaggregative E. coli (EAEC, 3 (1.5%) enteroinvasive E. coli (EIEC), and one 1(0.5%) enterohemorrhagic E. coli (EHEC) (Table 3).

cefotixime (26.7%), cotrimoxazole (26.7%) nalidixic acid (26.7%) and augmentin (6.7 %). High MICs90 (mg/L) (10. 12 to >128 ) were observed for the following examined drugs; ciprofloxacin, gentamycin, ceftriaxone and cefotaxime, respectively (Table 4).

The antibiotic susceptibility results of the 8 diarrheagenic E. coli isolates are shown in Table 4. The results has shown that most of 8 diarrheagenic E.coli isolates were susceptible to ciprofloxacin (100%), cefotixin (100%), gentamycin (100%), nalidixic acid (100%), amikacin (87.5%), nitrofurantoin (87.5%), ceftazidime (75%), ceftriaxone (75%), cefotixim (75%), augmentin (50%) and cotrimoxazole (37.5%) (Table 3). The susceptibility results of the 15 nondiarrheagenic E. coli isolates which were resistant to ≥ 3 drugs (Multidrug Resistant; MDR) were the following: cefotixin (73.3%), amikacin (53.3%), gentamycin (40%), nitrofurantoin (40%), ceftazidime (33.3%), ciprofloxacin (33.3%), ceftriaxone (26.7%),

The distribution results of bla CTX-M genes among 15 MDR non-diarrheagenic E.coli isolates were the following: CTX-M group I was found in 8(53.3%), CTX-M group II in 3(20%), CTX-M 15 in 9 (60%), CTX-M 8 and CTX-M 9 were not detected in any of the isolates, respectively (Table 5). While diarrheagenic E.coli results showed that CTX-M 8 and CTXM 9 were detected in 100%, CTX-M group II in 7 (87.5%), CTX-M group I in 3 (37.5%), and CTX-M 15 in 1 (12.5%) of the isolates, respectively ( Table 5). Both diarrheagenic and commensal E,coli carried only 47% and 50% of Class I integrons, respectively (Table 5). The distribution of potential quinolone resistance genes (Gyr A, Parc) and integrons among

Table 4. Antimicrobial susceptibility of 15 representative MDR of commensal E.coli and 8 diarrheagenic E. coli isolates from stool of children* Diarrheagenic E.coli

Non-diarrheagenic E.coli

Non-diarrheagenic E.coli

(%) Susceptible

(%) Susceptible

MICs90 (mg/L)

Ciprofloxacin

100

33.3

10.12

Cefotixin

100

73.3

-

Gentamycin

100

40

>64

Nalidixic acid

100

26.7

-

Amikacin

87.5

53.3

-

Nitrofurantoin

87.5

40

-

Ceftazidime

75

33.3

-

Ceftriaxone

75

26.7

>32

cefotaxime

75

26.7

>128

Augmentin

50

6.7

-

Cotrimoxazole

37.5

26.7

-

Antibiotics

*MDR accounted for 64 % of all 290 E.coli isolates © Under License of Creative Commons Attribution 3.0 License

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2014 Vol. 4 No. 1:2 doi: 10.3823/744

Table 5. Distribution of integrons, genes of bla(CTX-M) , and fluoroquinolone resistance (Gyr A and Par C) in diarrheagenic E.coli and multidrug resistant of commensal E.coli isolates No.(%) of 8 Diarrheagenic E.coli isolates

No.(%) of 15 Multidrug resistant commensal E.coli isolates

Genes

3 (37.5)

8 (53.3)

CTX-M I

7 (87.5)

3 (20)

CTX-M II

8 (100)

Null

CTX-M 8

8 (100)

Null

CTX-M 9

1 (12.5)

9 (60)

CTX-M 15

4(50)

7 (47)

Class 1 integrons

8 (100)

11 (73.3)

Gyr A gene

7 (87.5)

11 (73.3)

Par c gene

MDR commensal E.coli isolates were the following: 11/15 (73.3%) carried both Gyr A, Parc genes and 7/15 (47% ) harbored Class 1 integrons, whereas 8/8 (100%), 7/8 (87.5%) and 4/8(50%) of diarrheagenic E.coli isolates carried Gyr A, Parc genes and integrons, respectively.

Discussion This study demonstrates that the majority of children with watery or bloody diarrhea (62%) were aged less than1-year and presented with fever. These features are similar to many other studies described from the developing countries [1, 6-8-11]. The present study showed that diarrheagenic E.coli isolates were found only in 4 % of children with diarrhea and no single diarrheagenic E.coli isolate was detected in the control children group. The study also indicated the presence of (EAEC), (EIEC) and (EHEC), whereas the two types (EPEC and ETEC) were not detected in any child (Table 3). The presence rate of EAEC among diarrheal cases in this study is much similar to studies recently reported

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from Libya, Egypt and Iran [5,6,16-17], but in contrast to the other studies carried out in Kuwait and Jordan which had shown that EAEC was not a cause of diarrhea in their examined pediatric population [8,11]. However, the absence or low occurrence rate of EIEC (1.5%) in diarrhea cases and controls in this study is similar to the results of other reported studies from Egypt, Kuwait and Jordan [6,8,11]. In addition, it is important to note here that about half of the examined children were on antibiotic treatment during collection of their stool samples, and this might has been interfered with rate of recovery of diarrheagenic E.coli. The results of this study showed high rates of antimicrobial resistance (24-93%) to many antimicrobial classes, including 3-generation cephasporines (Ceftazidim, ceftriaxone, cefotixime ), and MDR E.coli accounted for 64% of all isolates (Table 4). All MDR commensal intestinal and diarrheagenic E.coli isolates carried various resistance rates (12100%) bla(CTX-M) genes, and particularly CTXM15 found more frequently in commensal intestinal E.coli than diarrheagenic E.coli isolates (60% versus 12.5%). These results are similar to some extend




to many recent studies carried out in other Arabic countries [15,18-21]. Over the past ten years, the bla(CTX-M) genes have become the most prevalent ESBLs among multidrug resistant organisms worldwide, started first in certain countries of Europe and South America, and later spread to India China, and other countries[ 15, 18,19, 21,23, 24]. In addition, this study showed high rates of Class I integrons in both MDR intestinal commensal E.coli and diarrheagenic E.coli isolates (73-100%) which carried one or �� both potential fluroq�� uinolone resistance genes (gyrA and parC) (Table 5). It has been previously established that high prevalence of Class I integrons contributes to increase the development of MDR in Enterobacteriaceae and particularly in fecal E.coli [25- 27]. Also, fluoroquinolone resistance in E. coli represents a substantial threat among community patients, since these drugs are increasingly used in empirical therapy of urinary tract and other infections [28-29]. In conclusion, diarrheagic E.coli types are not widely detected as cause of diarrhea in Libyan children of Benghazi , whereas high rates of normal intestinal E. coli isolates from children were MDR, carried blaCTX-M , Class I integrons and potential fluroq�� uinolone resistance genes. �� This study confirms the urgent need to monitor antimicrobial resistance trends of commensal intestinal E. coli in Libya.

Declaration of interest Nothing to declare.

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