INTRODUCTION:

E. Coli Is a Gram Negative, Facultatively Anaerobic, Rod-shaped, Commonly Found in The Intestines A Leading Cause of UTI’s, Is Becoming Resistant to Some Antibiotics Beta-lactamases Are Enzymes Produced by Bacteria That Provide Multi-Resistance To Β-lactam Antibiotics Such as Penicillins, Cephamycins, And Carbapenems ESBLs Are Enzymes That Mediate Resistance to Extended-spectrum (Third Generation) Cephalosporins (E.G., Ceftazidime, Cefotaxime, And Ceftriaxone) And Monobactams (E.G., Aztreonam) But Do Not Affect Cephamycins (E.G., Cefoxitin And Cefotetan) Or Carbapenems (E.G. Meropenem Or Imipenem)[1, 2]

These plasmid-mediated enzymes mostly evolved via point mutations of the classical TEM-1 and SHV-1 b-lactamases but other groups are increasingly prominent, notably the CTX-M types, which evolved via the escape and mutation of chromosomal b-lactamases from Kluyvera spp.[3]

Recently, there has been a notable surge in infections by extended spectrum beta-lactamase (ESBL)-producing Escherichia coli, which considerably limits treatment options.[4, 5]

In this study, we attempted to determine the detection frequencies of ESBLs in consecutive samples of clinical isolates of multidrug-resistant E. Coli with the various plate-screening methods mentioned above.

MATERIAL AND METHODS:

A total of 236 clinical isolates of E. coli were nonrepetitively and consecutively obtained from clinical specimens in the Al- Assad Teaching Hospital, Latakia, Syria, from October 2014 to November 2016.

Isolates came from six sources blood, urine, injury swab, vaginal swab, catheter swab and drain swabs. As showed in table 1.

Isolates were from different genders and inpatients and out patients. Samples were stored on Glycerol 20% tubes in ultra-freezer temperature -50°Cuntil sampling completed, methodology was according to CLSI.[6]

Table 1: Types and amounts of Isolates

Sample source	Count % (total n=236)
Urine	162 (68.6%)
Injury swab	33 (14%)
Blood	14 (5.9%)
Catheter swab	11 (4.7%)
Drain swab	10 (4.2%)
Vaginal swab	6 (2.5%)

Antimicrobial susceptibility testing and screening for ESBLs:

Routine antibiograms were determined by the disk diffusion method on Mueller-Hinton (MH) agar. Results were interpreted according to the CLSI (formerly NCCLS) standard and EUCAST (European Committee on Antimicrobial Susceptibility Testing).[7]

ESBL Disc Confirmation:

ESBL detection involves two important steps according to the latest recommendation from CLSI and EUCAST. The first is a screening test with an indicator cephalosporin which looks for resistance or diminished susceptibility, thus identifying isolates likely to be harboring ESBLs. The second step is a confirmation test (DDST) which evaluates the synergy between an oxyimino cephalosporin and clavulanic acid, distinguishing isolates with ESBLs from those that are resistant for other reasons. Figure 1 shows a screening test.

Double-Disc Synergy Test (DDST):

Discs containing cephalosporin (cefotaxime or ceftriaxone, ceftazidime, cefepime) were applied next to a disc with clavulanic acid, amoxicillin + clavulanic acid. Positive result is indicated when the inhibition zones around any of the cephalosporin discs are augmented in the direction of the disc containing clavulanic acid. The distance between the discs is critical and 20 mm center-to-center has been found to be optimal for cephalosporin 30 μg discs; however it was reduced (15 mm) or expanded (30 mm) for strains with very high or low resistance level, respectively.[8, 9] Figure 2 shows a positive DDST with the champagne cork or keyhole phenomenon.[10]

Statistical analysis:

Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS Inc, Chicago, IL) version 19.0. Univariate c2 and multivariate logistic regression analysis were performed to explore signiﬁcant differences and potential risk factors for ESBL-producing E coli infections. The difference was considered statistically signiﬁcant if P values were <.05.

Fig 1: Screening test CPD, CAZ and CTX are R, DDST positive (cefpodoxime Ceftazidime and cefotaxime)

Fig 2: Positive DDST

RESULTS:

During the study period, a total of 236 E. coli samples was isolated, by screening test we found that a total of 75 isolates (32%) were resistant for 3^rdgeneration cephalosporins. Susceptibility phenotypes of tested isolates are summarized in the antibiogram shown in Figure 1.

Table 2: Antibiotic susceptibility patterns of studied E coli isolates

Sample source	AUG %	CAZ %	CRO %	AZT %	CTX %	CPD %
Blood Culture	0	14	14	7	7	7
Catheter Swab	27	36	36	27	36	36
Urinary Culture	5	15	15	4	12	13
Injury Swab	24	58	64	39	58	55
Vaginal Swab	33	17	33	17	17	33
Drain Swab	10	60	50	20	30	30

(CPM Cefpodoxime, CTX Cefotaxime, CAZ Ceftazidime, CRO Ceftriaxone, AZT Aztreonam, GEN Gentamicin, AUG Augmentin)

Conﬁrmatory double disc synergy diffusion test showed that 26% of isolates were ESBL producers. Susceptibility to Augmentin was the most while resistance to Ceftriaxone was so common.

While studying the relation between resistance to antibiotics and sample source, there was highly signiﬁcant relation (P < .0001). Most MDR strains came from injury swabs 63%. Table 3 contains the details of MDR for each sampling source.

Table 2: details of MDR for each sampling source.

Sample source	MDR
Urine	16.7%
Injury swab	63%
Blood	14.2%
Catheter swab	36%
Drain swab	60%
Vaginal swab	33%

The percentage differences between inpatients and out patients was highly significant, total inpatients were 123 while outpatients were 113.

Only 3% of inpatients had ESBL positive, while 48% of inpatients were positive ESBL producers.

95% of MDR with ESBL producing isolates came from inpatients (P < .0001).

_{Figure 1 demonstrates the percentage relation of ESBL and patient Housing}

Fig3: ESBL and patient Housing

There was a significant relation between resisting to an antibiotic and sample source (P < .0001). CRO and CAZ were most resisted especially in urinary samples.

Table 2: Number of resistant sample to each antibiotic for each source

Sample source	AUG	CAZ	CRO	AZT	CTX	CPD
Blood Culture	0	2	2	1	1	1
Catheter Swab	3	4	4	3	4	4
Urinary Culture	8	24	25	7	19	21
Injury Swab	8	19	21	13	19	18
Vaginal Swab	2	1	2	1	1	2
Drain Swab	1	6	5	2	3	3

We noticed some Changes in appearance in some resistant colonies, some studies stated the same incidence[11]

DISCUSSION:

The results of this study showed a relatively high Prevalence of antibiotic resistant mostly because of the production of ESBLs in the samples of E. coli isolates examined collected from a Teaching hospital.

Our findings indicate the predominance of ESBL resistance type in E. coli compared to other types of resistance. Existence of high drug resistance to multiple antibiotics in E. coli in this study and several other related reports points towards negligence on patient’s part, incomplete treatment schedules, antibiotics misuse, self-prescription, misprescription, lack of regional antibiogram data, and limited knowledge about multidrug-resistant isolates and antimicrobial resistance among clinicians.

In our study, we used the latest CLSI and EUCAST recommendations and guidelines to detect ESBLs and the modified methods for eliminating false positive by using not only cefotaxime in confirmatory test and false negative results by adding cefepime to the screening test[6-9]

Prevalence of ESBL in Assad hospital in Latakia, which is a relatively safe city compared to other cities in Syria after the crisis, is 26% close to the results of ESBL tests conducted in the University Hospital Frankfurt, Germany on the Syrian refugees at the time of arrival to Germany in January 2016 which was 23.8% ESBL in E. Coli Isolates.[12]compared to a recent WHO study conducted in refugee camp southern Syria which had an astonishing high rate of ESBL 78% (95% were harbored by E.coli), these high rates are a normal result of living in a refugee camp for 6 years under very unhealthy surroundings[13]

The change in the form of some E. coli colonies which became a bit mucus like Klebsiella spp. colonies can be explained that Klebsiella strains are the source of resistant genes which moved to E. Coli strains through horizontal gene transfer and other genes responsible for the shape of the colony were accidentally transferred.

Inpatients were harboring 95% of total MDR strains which confirms it’s a nosocomial infection with the fact that the highest source of MDR was Injury and Catheter swabs, thus we recommend healthcare professionals and nurses to be more careful and strict to hygiene rules and personal hygiene.

In conclusion, we recommend adding this standard, cheap ESBL detection method to the routine microbial susceptibility tests run in Syrian hospitals in order to determine and isolate the sources of MDR infections and do some effort to contain and eliminate those strains.

Syrian health authorities need to practice proper control over the use of antibiotics to limit the spread of antibiotic misuse and therefore the spread of antibiotic resistance, and to ban selling antibiotics without prescription.

We also do recommend performing a PCR test to determine the molecular type for the resistant strains and the genes responsible for the spreading of this resistance.

REFERENCES:

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2. Wilcox, M.H., The tide of antimicrobial resistance and selection. Int J Antimicrob Agents, 2009. 34 Suppl 3: p. S6-10.

3. Jarlier, V., et al., Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis, 1988. 10(4): p. 867-78.

4. Coates, A.R.M., Antibiotic resistance. 2012, Heidelberg; New York: Springer.

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7. EUCAST, European Committee on Antimicrobial Susceptibility Testing Disk Diffusion Test Methodology. 2017.

8. EUCAST, EUCAST guideline for the detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. 2013.

9. Patel, J.B., Clinical, and I. Laboratory Standards, Performance standards for antimicrobial susceptibility testing. 2016.

10. Drieux, L., et al., Phenotypic detection of extended-spectrum beta-lactamase production in Enterobacteriaceae: review and bench guide. Clin Microbiol Infect, 2008. 14 Suppl 1: p. 90-103.

11. Olorunmola, F.O., D.O. Kolawole, and A. Lamikanra, Antibiotic Resistance and Virulence Properties in Escherichia Coli Strains from Cases of Urinary Tract Infections. Afr J Infect Dis, 2013. 7(1): p. 1-7.

12. Reinheimer, C., et al., Multidrug-resistant organisms detected in refugee patients admitted to a University Hospital, Germany JuneDecember 2015. Euro Surveill, 2016. 21(2).

13. Kassem, D.F., et al., Multidrug-Resistant Pathogens in Hospitalized Syrian Children. Emerg Infect Dis, 2017. 23(1): p. 166-168.

Received on 10.03.2017 Modified on 28.06.2017

Research J. Pharm. and Tech. 2017; 10(7): 2433-2436.

DOI: 10.5958/0974-360X.2017.00430.9