Characterization of ESBLs and Associated Quinolone Resistance in Escherichia coli and Klebsiella pneumoniae Isolates from an Urban Wastewater Treatment Plant in Algeria

Abstract

The aim of the study was the characterization of extended spectrum beta-lactamases (ESBLs) and quinolone resistance in cefotaxime-resistant coliform isolates from a wastewater treatment plant (WWTP). ESBLs were detected in 19 out of 24 isolates (79%) from raw water and in 21 out of 24 isolates (87.5%) from treated water, identified as Klebsiella pneumoniae and Escherichia coli. Molecular characterization of ESBLs and quinolone resistance showed allele profiles CTX-M-15 (3), CTX-M-3 (5), CTX-M-15+qnrB1 (1), CTX-M-3+qnrB1 (1), CTX-M-15+aac-(6′)-Ib-cr (4), and CTX-M-15+qnrB1+aac-(6′)-Ib-cr (7). A double mutation S83L and D87N (GyrA) and a single mutation S80I (ParC) were detected in ciprofloxacin-resistant E. coli isolates. In K. pneumoniae, mutations S83I (GyrA)+S80I (ParC) or single S80I mutation were detected in ciprofloxacin-resistant isolates, and no mutation was observed in ciprofloxacin-susceptible isolates. bla_CTX-M, qnrB1, and aac-(6′)-Ib-cr were found, respectively, in these genetic environments: ISEcp1-bla_CTX-M-orf477, orf1005-orf1-qnrB1, and Tn1721-IS26-aac-(6')-Ib-cr-bla_OXA-1-catB4. bla_CTX-M-15 was located on IncF plasmid in E. coli and bla_CTX-M-3 on IncL/M plasmid in both species (E. coli and K. pneumoniae). E. coli isolates were affiliated to the phylogroups/MLST: D/ST405 (CC405), A/ST10 (CC10), A/ST617 (CC10), and B1/ST1431. K. pneumoniae isolates belonged to phylogroup KpI and to sequence types ST15, ST17, ST36, ST48, ST54, and ST147. The study showed a multi-drug resistance at the inflow and outflow of the WWTP, with ESBL production, plasmid-mediated quinolones resistance, and mutations in topoisomerases. The findings highlight the similarity of antibiotic resistance mechanisms in the clinical setting and the environment, and the role of the latter as a source of dissemination of resistance genes.

Introduction

The indiscriminate use of antibiotics has contributed to their significant presence in the environment, promoting resistant microorganisms by selection, mutation, and recombination.^4,12,30 Contaminated aquatic environments are instrumental in dissemination of resistant bacteria (resistance genes) that can reach humans through direct contact or food chains.^12,50 The assessment of antibiotic resistance and elucidation of its mechanisms in human-related environments are an invaluable complement to clinical studies, for the understanding of the development and spread of antimicrobial resistance. The wastewater treatment plant (WWTP) is an important link in the recycling of water, it constitutes a site of conjunction and concentration of various types of pollution, and its effluents have a significant impact on sanitary quality of the environment. The analysis of microbial antibiotic resistance at the level of WWTP is interesting, because these data are representative of the evolution that might occur in the aquatic environment. Our study was focused on the beta-lactams and quinolones resistance because of the major importance of these antibiotic classes in the anti-infective therapy. One of the most efficient mechanisms of resistance to broad-spectrum cephalosporins is the production of extended spectrum beta-lactamases (ESBL), which are plasmid-encoded clavulanate-susceptible enzymes conferring resistance to third- and fourth-generation cephalosporins and to monobactams.^21,37 ESBLs are a major public health problem, as they are associated with outbreaks with a significant cause of morbidity and mortality. Until the late 1990s, detected ESBLs were derived from the old narrow-spectrum penicillinases TEM-1, SHV-1, and SHV-2. Subsequently, other types of ESBLs have emerged as PER, VEB, GES, and CTX-M. Currently, CTX-M are the most prevalent ESBLs, and their dissemination is fast and worldwide.^21,37 Other resistance genes, in particular plasmid-mediated quinolone resistance (PMQR) determinants (qnr, aac(6′)-Ib-cr, and qepA), may be associated with ESBLs.^24,45 Qnr proteins belong to the pentapeptide repeat family that directly protect DNA topoisomerases from quinolone inhibition. The enzyme AAC(6′)-Ib-cr is a variant of common aminoglycoside acetyltransferase AAC(6′)-Ib that is able to N-acetylate piperazinyl substituent of ciprofloxacin and norfloxacin. QepA protein is an efflux pump related to major facilitator superfamily transporters.⁴⁵ The level of resistance conferred by PMQR determinants is low, but they can facilitate the emergence of bacteria with a higher resistance level.^37,45

Although many studies on antibiotic resistance in the environment have been performed, including at WWTPs, more research on the subject is needed. The knowledge of the mechanisms, resistance genes, resistance-mediating mutations, mobile genetic elements, and phylogeny of plasmids and of resistant strains is still insufficient, even more in Algeria and the African countries, where data related to the environment are almost nonexistent. The aim of the study was the detection and characterization of ESBLs and associated quinolone resistance in coliform isolates recovered at the level of an urban WWTP in Algeria.

Materials and Methods

Sampling and bacteria isolation

Samples of wastewater and treated water were collected at three different times (March 14, March 28, and April 4; 2010) at the inflow and outflow of a WWTP located beside Boumerdes city, a seaside town in the center of Algeria. The WWTP was designed for a population of approximately 75,000 people with capacity treatment of 15,000 m³·day⁻¹. It mostly treats domestic wastewater, including a low amount of hospital and industrial wastes. The treatment is a biological process with activated sludge using aeration tanks and clarifiers. The treated wastewater is discharged directly without any further treatment in Tatareg River, at 3 Km upstream from the sea. Microbiological and physico-chemical analysis of the raw water and treated water showed that microbial populations (total flora, total coliforms, and thermotolerant coliforms), chemical oxygen demand, and biological oxygen demand decreased after treatment, with reductions ≥94% and of approximately 99%, respectively (Table 1). These results conform to recommended quality standards and are indicative of efficiency of treatment.

Table 1.

Physico-Chemical and Microbiological Parameters of Raw and Treated Water of Wastewater Treatment Plant

	Raw water sample			Treated water samples			Efficiency removal (%)
Parameters	S1	S2	S3	S1	S2	S3	S1	S2	S3
Total flora (CFU·100 ml⁻¹)	3.1×10⁷	1.64×10⁷	2.4×10⁸	3.8×10⁴	3.82×10⁴	1.63×10⁵	99.8	99.7	99.9
Total coliforms (CFU·100 ml⁻¹)	2.08×10⁶	2.84×10⁶	1.0×10⁷	9.2×10³	1.19×10⁴	1.98×10⁴	99.5	99.5	99.8
Fecal coliforms (CFU·100 ml⁻¹)	1.19×10⁵	1.54×10⁴	5.6×10⁴	5.0×10³	8.9×10²	1.06×10³	95.7	94.2	98
BOD (mgO₂·L⁻¹)	210	240	300	04	02	02	98	99	99.3
COD (mg O₂·L⁻¹)	700	780	790	12	8	8	98.2	98.9	98.9

S1, S2, and S3: first, second, and third sampling, respectively.

COD, chemical oxygen demand; BOD, biochemical oxygen demand.

A volume of 100 ml of serial decimal dilutions was filtered on cellulose nitrate membranes of 0.45 μm pore size (Millipore). Filters were placed onto tergitol-7 agar, a coliform selective medium, supplemented or not with cefotaxime at a concentration of 2 μg·ml⁻¹ and incubated overnight at 37°C.

To compare cefotaxime resistance rates in influents and effluents of WWTP, a Chi-squared test was used and the difference was considered significant when p was less than 0.05.

A total of 48 cefotaxime-resistant colonies, including 24 from raw water and 24 from treated water, were picked and screened for ESBL production.

Screening, identification, and molecular typing of ESBL-producing isolates

Cefotaxime-resistant isolates were screened for ESBL production by using the Double-Disc Synergy Test (DDST).³¹ The identification of DDST-positive isolates was done with API 20E (Biomerieux). The clonal relationship between ESBL-producing isolates was analyzed by Enterobacterial repetitive consensus PCR (ERIC-PCR) using primer ERIC2 as previously described.³¹ Fingerprints were visually compared, and the patterns differing by at least one amplification band were classified as different.

Antibiotic resistance testing

Antibiotic resistance profiles of ESBL-producing isolates were determined by the disk diffusion method on Mueller–Hinton agar, according to the recommendations of the Antibiogram Committee of the French Society for Microbiology⁸ (www.sfm-microbiologie.org). The following antibiotic disks (Bio-Rad) were used (μg or International Unit “IU”/disk): cefotaxime 30 μg; ceftriaxone 30 μg; aztreonam 30 μg; ceftazidime 30 μg; cefoxitin 30 μg; cefepime 30 μg; cefpirome 30 μg; imipenem 30 μg; kanamycin 30 IU; gentamicin 15 μg; sulfonamides 200 μg; tetracyclines 30 IU; nalidixic acid 30 μg; ciprofloxacin 5 μg; chloramphenicol 30 μg; and rifampicin 30 μg. Escherichia coli ATCC 25922 was used as a control strain for antimicrobial susceptibility testing.

MICs of cefotaxime, ceftazidime, cefepime, cefoxitin, imipenem, and ciprofloxacin were determined by agar-dilution method and interpreted according to the guidelines of CA-SFM.⁸

Characterization of ESBL and quinolone-resistance encoding genes

The identification of β-lactamase genes was carried out by PCR using universal primers for CTX-M and TEM, and specific primers for CTX-M groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) and OXA-1, as previously described.^1,40 PCR-obtained products were sequenced and analyzed with the BLAST and FASTA programs of the National Center for Biotechnology Information (www.ncbi.nlm.nhi.gov).

qnrA, qnrB, and qnrS genes were detected by multiplex PCR, and qnrC, qnrD, aac-(6′)-Ib, and qepA were detected by simplex PCR using specific primers.^15,40 Quinolone-resistance-determining regions (QRDR) of gyrA and parC genes were amplified as previously described.⁴² PCR products were sequenced, and nucleotide sequences and deduced-protein sequences were analyzed.

Detection of class 1 integrons and genetic environment of ESBL and PMQR genes

Class 1 integrons were detected by multiplex PCR targeting intI1, sulI1, and qacΔE genes.³¹ The variable region between 5′- and 3′-conserved segments was characterized by PCR and sequence analysis using primers 5′Cs and 3′Cs.³¹

The genetic association of ISEcp1 and orf477 sequences with bla_CTX-M genes was investigated by PCR using primers PROM+/CTXM-A2¹⁶ and CTXM3int/orf1pol.⁴⁰ The spacer region between ISEcp1 and bla_CTX-M genes was analyzed by sequencing the PCR product obtained with primers CTX-M3GF/R.¹

The presence of orf1005 sequence upstream qnrB1 gene was screened by PCR and sequence analysis using primers IS3000R and QnrBnewR.⁴²

The genetic environment of aac-(6′)-Ib-cr was investigated by PCR and sequence analysis using primer combinations Tn1721-F2/aac-Ib-R and aac-Ib-F/catB3-R.⁴² The presence of aac-(6′)-Ib-cr in a class 1 integron was screened using primer combinations RV1-F/aac-Ib-R and aac-Ib-F/RV1-R.⁴²

Conjugation experiments and plasmid analysis

Mating assays were performed as previously described using sodium azide resistant E. coli BM21 as a recipient.³ Transconjugants were selected on brain heart infusion agar supplemented with cefotaxime (4 μg·ml⁻¹) and sodium azide (250 μg·ml⁻¹), and subjected to antibiotic resistance testing, DDST, PCR, and sequence analysis.

The determination of the incompatibility group of plasmids carrying ESBL and/or PMQR determinants was performed by PCR-based replicon typing method as previously described.^6,42

PFGE gels of genomic DNA digestion with S1 nuclease were analyzed by Southern blot hybridization using CTX-M, IncL/M, and IncF probes (PCR Dig Probe Labelling Mix; Roche Applied Science, Barcelona, Spain).⁴²

Phylogenetic group and multilocus sequence typing

E. coli phylogenetic group was done by PCR based on chuA and yjaA genes, and the DNA fragment TspE4.C2, as previously described.¹⁰ Klebsiella pneumoniae phylogenetic group was determined by RFLP of gyrA gene using the restriction enzymes Taq1α and Hae III.⁴²

Multilocus sequence typing (MLST) was performed on E. coli and K. pneumoniae CTX-M-producing isolates, according to the previously described protocols available at http://mlst.ucc.ie/mlst/dbs/Ecoli and www.pasteur.fr/recherche/genopole/PF8/mlst, respectively.

Results

The rates of cefotaxime-resistant coliforms in relation to total coliforms were 0.22% (n=4.57×10³ CFU·100 ml⁻¹)±0.05, 0.97% (n=2.75×10⁴ CFU·100 ml⁻¹)±0.11 and 1.62% (n=1.62×10⁵ CFU·100 ml⁻¹)±0.19 for the three samples of raw water obtained before treatment, and 14.45% (n=1.32×10³ CFU·100 ml⁻¹)±0.9, 8.23% (n=9.79×10² CFU·100 ml⁻¹)±0.8, and 2.57% (n=5.08×10² CFU·100 ml⁻¹)±0.24 for those of treated water. The statistical analysis of resistance rates showed their highly significant increase after treatment (p=0.00003).

The detection of production of extended-spectrum β-lactamases was performed on 48 cefotaxime-resistant coliforms, divided into 24 isolates obtained before and after treatment. Forty isolates (83.3%) were DDST positive, 19 isolates (79%) (K. pneumoniae n=11 and E. coli n=8) from raw water, and 21 isolates (87.5%) (K. pneumoniae n=20 and E. coli n=1) from treated water.

Other than resistance to cefotaxime (MICs: 4 mg·L⁻¹ to 128 mg·L⁻¹), antibiotic susceptibility testing of the 40 DDST-positive isolates showed high percentages of resistance to other beta-lactams: ceftazidime (75%, MIC: ≤4 to 128 mg·L⁻¹), aztreonam (77.5%), cefpirome (80%), and cefepime (75%, MIC: ≤4 to 128 mg·L⁻¹). Nevertheless, all isolates showed susceptibility to imipenem (MIC: ≤2 mg·L⁻¹) and susceptibility or intermediate resistance to cefoxitin (MIC: 4 to 32 mg·L⁻¹). With regard to other antibiotic families, a marked resistance was observed to quinolones (nalidixic acid: 87.5% and ciprofloxacin: 85%), rifampicin (70%), and sulphonamides (65%), and moderate resistance was noted to aminoglycosides (gentamicin: 27.5% and kanamycin: 7.5%), tetracycline (25%), and chloramphenicol (10%).

PCR detection of beta-lactamase genes showed bla_CTX-M gene in the 40 DDST-positive isolates, bla_TEM in 38 isolates, and bla_OXA-1 in 27 isolates. Multiplex PCR screening for PMQR determinants showed that 23 isolates contained qnrB gene and 29 isolates carried aac-(6')-Ib gene. All isolates were negative for genes qnrA, qnrS, qnrC, qnrD, and qepA.

The search of class 1 integrons by multiplex PCR in the 40 CTX-M-producing isolates revealed their presence in 35 isolates (87.5%). intI1, sul1, and qacEΔ1 genes were detected in 31 isolates (5 E. coli and 26 K. pneumoniae), while only intI1 gene was detected in 4 isolates (1 E. coli and 3 K. pneumoniae). The amplification of the variable region of class 1 integrons was positive for 22 isolates (17 K. pneumoniae and 5 E. coli), with the size being 800 bp in 2 E. coli, 1,000 bp in 1 K. pneumoniae, 2,000 bp in 3 E. coli, and 2,500 bp in 16 K. pneumoniae.

Molecular typing of 40 CTX-M-producing isolates by ERIC-PCR showed 4 genetic profiles among the 9 isolates of E. coli and 9 profiles among the 31 isolates of K. pneumoniae.

On the basis of their resistance phenotypes (resistance profiles and MICs) and genotype (ERIC-PCR profile, resistance genes, integrons, insertion sequences, and spacer region), 21 isolates (6 E. coli and 15 K. pneumoniae) were selected for sequence analysis of β-lactamase and PMQR genes. Selected strains harbored the following genes: seven isolates CTX-M+qnrB+aac-(6′)-Ib, five isolates CTX-M+aac-(6′)-Ib, two isolates CTX-M+qnrB, and seven isolates CTX-M. The allele profiles detected were as follows: CTX-M-15 (two E. coli and one K. pneumoniae), CTX-M-3 (two E. coli and three K. pneumoniae), CTX-M-15+qnrB1 (one K. pneumoniae), CTX-M-3+qnrB1 (one E. coli), CTX-M-15+aac-(6′)-Ib-cr (one E. coli and three K. pneumoniae), and CTX-M-15+qnrB1+aac-(6′)-Ib-cr (seven K. pneumoniae) (Table 2).

Table 2.

Resistance Phenotype and Genotype of Extended Spectrum Beta-Lactamase-Producing Isolates Recovered from Raw and Treated Water of Wastewater Treatment Plant

				MIC (mg/L)										QRDR mutation
Isolates	ERIC profile	Phylogeny	ST (CC)	CTX	CAZ	FEP	FOX	IMP	CIP	Additional antibiotic resistance	Detected genes of integrons1 (size of variable region)	CTX-M genetic environment; other β-lactamases	Genetic environment of PMQR determinants	GyrA	ParC	Plasmid replicon type (size kb)
Isolates from raw water
Ec 1	E3	A	10 (10)	4	≤4	≤4	16	≤2	32	ATM-CPO-NA-TE-SSS	F (800 bp)	ISEcp1-W- bla _CTX-M-3-orf477	orf1005-orf1- qnr B1	S83L D87N	S80I	F-L/M (84.5)-FIB
Ec 2	E3	A	10 (10)	64	8	16	16	≤2	32	ATM-CPO-NA-TE-SSS	F (800 bp)	ISEcp1-W- bla _CTX-M-3-orf477	—	S83L D87N R91L	S80I	F
Ec 3	E2	A	617 (10)	64	16	8	16	≤2	>32	ATM-CPO-NA-TE-SSS-C-RA	F (2 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_OXA-1	Tn1721-IS26- aac(6 ′ )Ibcr -bla_OXA-1-catB4	S83L D87N	S80I	F(155)-I1
Ec 4	E2	D	405 (405)	64	16	16	16	≤2	>32	CPO-NA-TE-SSS-K-RA	IntI1 (—)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1	—	S83L D87N A93E	S80I	F
Ec 5	E1	D	405 (405)	64	16	8	16	≤2	>32	NA	— (—)	ISEcp1-W- bla _CTX-M-15-orf477	—	S83L D87N	S80I	F
Kp 1	K1	KpI	54	64	4	16	4	≤2	≤0.062	—	— (—)	ISEcp1-VW- bla _CTX-M-3-orf477	—	wild	wild	L/M
Kp 2	K2	KpI	17	32	4	8	4	≤2	≤0.062	CPO	— (—)	ISEcp1-VW- bla _CTX-M-3-orf477	—	wild	wild	L/M ^T(80)
Kp 3	K4	KpI	36	64	64	64	4	≤2	0.25	—	— (—)	ISEcp1-VW- bla _CTX-M-3-orf477	—	wild	wild	L/M
Kp 4	K3	KpI	147	64	16	8	4	≤2	≤0.062	TE-SSS-RA	F (—)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1	orf1005-orf1- qnr B1	wild	wild	ND
Kp 5	K5	KpI	17	64	64	64	16	≤2	≤0.062	ATM-CPO-NA-SSS-RA	F (—)	ISEcp1-VW- bla _CTX-M-15-orf477	—	wild	wild	ND
Kp 6	K6	ND	ND	128	128	128	16	≤2	32	ATM-CPO-NA-SSS-RA	F (2.5 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND
Kp 7	K6	KpI	147	128	128	128	4	≤2	>32	ATM-CPO-NA-SSS-GN-RA	F (2.5 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	orf1005-orf1- qnr B1 Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	wild	S80I	ND
Kp 8	K8	KpI	15	64	16	8	8	≤2	8	ATM-CPO-NA-TE-SSS-K	F (1 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	wild	S80I	ND
Kp9	K4	KpI	48	128	128	32	4	≤2	0.125	ATM-CPO-TE-RA	IntI1(—)	ISEcp1-W- bla _CTX-M-15-orf477; bla_OXA-1	orf1005-orf1^- qnr* B1 Tn1721-IS26- aac(6 ′ )Ibcr - bla_OXA-1-catB4	wild	wild	ND
Isolates from treated water
Ec 6	E4	B1	1431	64	16	64	32	≤2	32	ATM-CPO-NA-TE	F (2 kb)	ISEcp1-W- bla _CTX-M-3-orf477	—	S83L D87N	S80I	I1-F^T-L/M^T(98.5)
Kp 10	K6	ND	ND	128	128	64	8	≤2	16	ATM-CPO-C-RA	F (2.5 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND
Kp 11	K6	KpI	147	128	128	128	8	≤2	>32	ATM-CPO-RA	F (2.5 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	orf1005-orf1- qnr B1 Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND
Kp 12	K6	ND	ND	128	128	128	8	≤2	>32	NA-SSS-GN-RA	F (2.5 kb)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	orf1005-orf1- qnr B1 Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND
Kp 13	K9	KpI	ND	128	128	128	8	≤2	>32	ATM-CPO-NA-SSS-GN-RA	F (—)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	orf1005-orf1- qnr B1 Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND
Kp 14	K6	ND	ND	128	128	128	16	≤2	>32	ATM-CPO-NA-SSS-C-RA	F (—)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	orf1005-orf1- qnr B1 Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND
Kp 15	K6	KpI	ND	128	128	64	8	≤2	>32	ATM-CPO-NA-GN-RA	F (—)	ISEcp1-W- bla _CTX-M-15-orf477; bla_TEM-1; bla_OXA-1	orf1005-orf1- qnr B1 Tn1721-IS26^- aac(6* ′ )Ibcr - bla_OXA-1-catB4	S83I	S80I	ND

truncated sequence.

self-transferable plasmid.

Plasmid carrying bla_CTX-M was underlined. Genes of CTX-M and PMQR are in bold.

Ec, Escherichia coli; Kp, Klebsiella pneumoniae; CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam; CPO, cefpirome; FEP, cefepime; FOX, cefoxitin; IMP, imipenem; CIP, ciprofloxacin; NA, nalidixic acid; TE, tetracycline; SSS, sulfonamides; GN, gentamicin; K, kanamycin; C, chloramphenicol; RA, rifampicin; ND, not determined, ST, sequence type; CC, clonal complex; F, presence of the three genes of class 1 integrons (IntI1, sul1, and qacΔE), IntI1: presence only of integrase gene.

The six E. coli isolates having a high level of resistance to ciprofloxacin (MIC ≥32 mg·L⁻¹) showed a double mutation S83L and D87N and a single mutation S80I in the quinolone resistance-determining region (QRDR) of genes encoding subunits GyrA and ParC of DNA gyrase and topoisomerase IV, respectively. In addition, a third amino-acid change R91L or A93E in GyrA was detected in isolates Ec2 and Ec4, respectively. In K. pneumoniae isolates, mutations S83I in GyrA and S80I in ParC were detected in five isolates (ciprofloxacin MIC ≥32 mg·L⁻¹) having, in addition, aac-(6')-Ib-cr and qnrB1 genes, and in two isolates (ciprofloxacin MIC of 16 to 32 mg·L⁻¹) having aac-(6')-Ib-cr. Two other isolates with only S80I mutation in ParC and aac-(6')-Ib-cr or qnrB1+aac-(6')-Ib-cr genes were resistant to ciprofloxacin (MICs: 8 mg·L⁻¹ and >32 mg·L⁻¹, respectively). Six ciprofloxacin-susceptible isolates (MIC ≤0.25 mg·L⁻¹), two of them having qnrB1 or qnrB1+aac-(6')-Ib-cr, did not show any mutation (Table 2).

An analysis of the genetic environment of bla_CTX-M of the 21 isolates showed the presence of ISEcp1 upstream and orf477 downstream of the gene (Table 2). The genetic linkage between bla_CTX-M genes and ISEcp1 was detected in 19 isolates, with amplification products of 1,000 bp for 2 CTX-M-3-producing E. coli and 13 CTX-M-15-producing K. pneunomiae isolates, and of 1,100 bp for 3 CTX-M-3-producing K. pneunomiae and 1 CTX-M-15-producing K. pneunomiae. The sequence analysis of the spacer region between bla_CTX-M and ISEcp1 revealed a size of 127 bp corresponding to (V+W) sequences¹⁶ (for amplicons of 1,100 bp) and of 48 bp corresponding to W sequence¹⁶ (for amplicons of 1,000 bp) in both CTX-M-3-and CTX-M-15-producing E. coli and K. pneumoniae (Table 2).

The association of qnrB1 with orf1005 encoding a putative transposase (pspA) was demonstrated in all qnrB1-carrying isolates by PCR amplification of fragments of 2 kb in eight isolates and 800 bp in one isolate. Sequencing of the 2 kb amplicons showed the presence of orf1 between orf1005 and qnrB1, which resembles a pspF gene coding for the transcriptional activator of the stress-inducible psp operon (Table 2).

A genetic environment analysis of aac-(6')-Ib-cr gene showed that, in both E. coli and K. pneumoniae, it was flanked by Tn1721 and IS26 upstream and bla_OXA-1 (oxacillin-hydrolizing capabilities) and catB4 (chloramphenicol resistance) downstream, forming the following structure: Tn1721-IS26-aac-(6')-Ib-cr-bla_OXA-1-catB4. However, in 10 out of 12 isolates carrying aac-(6')-Ib-cr, IS26 (50 bp) is truncated (Table 2). This analysis also allowed to locate the gene aac-(6')-Ib as gene cassette in a class1 integron in the following arrangement: aac-(6')-Ib-aadA1-bla_OXA-9.

PCR replicon typing of plasmids showed a common presence of plasmids IncL/M in isolates having bla_CTX-M-3, except E.coli Ec2. bla_CTX-M-3 was found transferable from two isolates (E. coli Ec6 and K. pneumoniae Kp2) in association with plasmids IncL/M. Southern hybridization confirmed the location of bla_CTX-M-3 on plasmids IncL/M of about 84.5 kb (E. coli Ec1), 98.5 kb (E. coli Ec6), and 80 kb (K. pneumoniae Kp2). bla_CTX-M-15 gene of E. coli Ec3 was located on IncF plasmid of about 155 kb (Table 2).

Phylogenetic group and MLST were conducted on 15 CTX-M–producing isolates (six E. coli and nine K. pneumoniae). E. coli isolates were affiliated to the following phylogroups, sequence types (ST), and clonal complexes (CC): D/ST405 (CC405), A/ST10 (CC10), A/ST617 (CC10), and B1/ST1431. K. pneumoniae isolates belonged to phylogroup KpI and to sequence types ST15, ST17, ST36, ST48, ST54, and ST147 (Table 2).

Discussion

The rates of cefotaxime-resistant bacteria in relation to total coliforms were significantly higher in the three treated water samples than in the raw water samples. This come in agreement with studies reporting an increase of bacterial resistance at outflow of WWTPs,^{18,19,20,47,52} mainly due to selective pressure and genetic exchange in sludge.^4,18,39 To study mechanisms involved in the β-lactams resistance, detection of extended-spectrum β-lactamases was performed on 48 cefotaxime-resistant coliforms. Results showed a high prevalence of ESBL-producing isolates, identified as K. pneumoniae and E. coli. This confirms the major role of ESBL in acquired resistance to broad spectrum β-lactams in Gram-negative bacilli, particularly in K. pneumoniae and E. coli.²¹ This finding is consistent with studies reporting the presence of ESBLs in aquatic environments in general and in WWTP in particular, before and after treatment.^{15,20,38,39,47} Percentages of ESBL-producing isolates were substantially the same in influent and effluent of WWTP, indicating that it is the same resistance mechanism which prevailed before and after treatment. However, ESBL-producing isolates of treated water were more resistant (frequency and MICs) against ceftazidime, fourth-generation cephalosporins (cefepime and cefpirome), and non beta-lactams (quinolones, aminoglycosides, chloramphenicol, and rifampicin). This corroborates our preceding results, showing an increase of resistance after treatment.

Class 1 integrons were detected in 35 out of 40 ESBL-producing isolates. This high prevalence is related to the role of these elements in the spread of antibiotic resistance in wastewater and surface water.¹⁹ However, no direct association with bla_CTX-M genes was found. This is consistent with what was reported for the cluster CTX-M-1.¹⁶

All ESBL belonged to CTX-M-1 cluster and often associated with TEM-1 and/or OXA-1; this association, frequently in the same plasmid, was commonly reported in clinical Enterobacteriaceae.^31,35 The sequence analysis of ESBLs of the 21 selected isolates showed a complete homology with CTX-M-3 (n=6) and CTX-M-15 (n=15) alleles. In general, the presence of CTX-M ESBLs in aquatic environments bacteria was reported.^9,15,20,26 With regard to the two alleles found in our study, CTX-M-15 was reported in Enterobacteriaceae from seawater of beach, rivers, and effluents of WWTP and of hospitals,^{1,14,15,20,48} and CTX-M-3 from rivers and effluent of WWTP.^20,48 The epidemiology of ESBL in clinical Enterobacteriaceae has evolved over the last decade with the worldwide explosive diffusion of CTX-M family, especially types CTX-M-3 and CTX-M-15, which are now the most widespread.^21,37 These findings on isolates from WWTP are consistent with studies in clinical settings in Algeria, reporting that CTX-M-15 and CTX-M-3 were predominant and endemic.^22,31,49 bla_CTX-M genes were found flanked upstream and downstream by insertion sequence ISEcp1B and orf477. This structure was previously described in clinical and environmental isolates,^16,48 and the high prevalence of ISEcp1 emphasizes its role in the expression and dissemination of these resistance genes.^16,36 Both sequences (W+V) or only W have characterized the spacer region between bla_CTX-M and ISEcp1 in our strains. This result does not correspond to what was previously reported in clinical strains, namely W+V for CTX-M-3 and W for CTX-M-15 ¹⁶ or V+W for both CTX-M-3 and CTX-M-15.³¹ This would suggest an evolution of environmental strains to resistance via genetic events different compared with clinical strains. In addition, the presence of sequence W (48 bp) as a spacer region for CTX-M-3 in E. coli was not previously described.

ESBL-producing bacteria are often described as co-resistant to other antibiotics, thereby enhancing their selection and persistence. A significant correlation between resistance to broad-spectrum cephalosporins and resistance to ciprofloxacin was already reported.^33,42 The screening for PMQR determinants among the 21 selected CTX-M-producing isolates showed the presence of qnrB1 genes in 1 CTX-M-3-producing E. coli and 1 CTX-M-15-producing K. pneumoniae, aac-(6')-Ib-cr in 4 CTX-M-15-producing isolates (one E. coli and three K. pneumoniae), and qnrB1+aac-(6')-Ib-cr in 7 CTX-M-15-producing K. pneumoniae. As has been described in clinical isolates, including in Algeria, qnrB1 and aac(6')-Ib-cr are most often associated with CTX-M-15.^24,42,45 qnrB1 and aac-(6')-Ib-cr were rarely described in the environment, respectively, only once in Germany⁴⁷ and twice in the Czech Republic and the USA,^15,25 in WWTPs. Upstream of qnrB1 was found an orf1005 encoding a putative transposase and between them there was orf1, which, except for one isolate, corresponds to the full sequence of pspF gene involved in regulation of the stress psp operon. This structure was reported by Jacoby et al.²⁴ in clinical isolates, however with a truncated orf1. In the genetic environment of aac-(6′)-Ib-cr, the structure “Tn1721-IS26-aac-(6′)-Ib-cr-bla_OXA-1-catB4” described by Ruiz et al.⁴² was found in our strains, with a truncated IS26 for the most K. pneumoniae isolates. aac(6′)-Ib gene was found as a gene cassette in class 1 integron in the following arrangement “aac-(6′)-Ib-aadA1- bla_OXA-9”, resembling, in part, what was previously described in K. pneumoniae.⁴⁴

In addition to PMQR determinants, the (S83L, D87N+S80I) mutation profile of QRDR of GyrA and ParC found in all E. coli explained their high resistance to ciprofloxacin (MIC ≥32 mg·L⁻¹). To our knowledge, the additional mutations R91L and A93E found in two strains of E. coli are described for the first time. Investigation on their impact on quinolone resistance would be interesting. Klebsiella isolates with prevalent double mutation S83I (GyrA) +S80I (ParC) showed a high level of resistance to ciprofloxacin, in particular with the concomitant presence of qnrB1 and aac-(6′)-Ib-cr. A single mutation S80I in ParC was observed in two isolates, with a high level of ciprofloxacin resistance of the isolate having qnrB1 and aac-(6′)-Ib-cr and a moderate resistance of the other with only aac-(6′)-Ib-cr. It is generally reported that mutations in ParC have little impact on the resistance to fluoroquinolones, except when they are associated with mutations in GyrA. It is known that topoisomerase IV is a secondary target of quinolones compared with DNA gyrase; therefore, mutations conferring resistance affect first DNA gyrase, particularly GyrA and then, in a second step, they concern ParC, resulting in a high level of resistance. In our case, resistance levels reached by our isolates might be attributed to additional PMQR genes; indeed, these latter are considered a way of strengthening the quinolone resistance, and their transcriptional level and copy number may affect this phenotype.^28,41 Finally, PMQR determinants alone did not confer resistance to ciprofloxacin. This is consistent with a previous report that S83L (GyrA) had the strongest influence on quinolone resistance,² and PMQR determinants facilitate the selection of mutants with high levels of resistance. Mutations D87N, S83L, and S80I are the most described, and (S83L, D87N,+S80I) mutation profile was the most prevalent in clinical and environmental quinolone-resistant Enterobacteriaceae.^42,46 However, as in our study, the mutation S83I (GyrA) can be prevalent among K. pneumoniae.⁵¹

bla_CTX-M-15 was located on a large IncF plasmid in E. coli and bla_CTX-M-3 on large IncL/M plasmids in both E. coli and K. pneumoniae. The transference by conjugation of IncL/M plasmids from two isolates (one E. coli and one K. pneumoniae) in addition to bla_CTX-M-3 was demonstrated. This finding is in accordance with the location of bla_CTX-M-15 on IncF plasmids reported in a study on multiresistant E.coli and K. pneumoniae from effluents of WWTP in Czech Republic;¹⁵ while it does not match with previous studies on clinical and environmental Enterobacteriaceae in Algeria reporting bla_CTX-M-15 on IncL/M and IncI1 plasmids, respectively.^1,23,32 In general, bla_CTX-M-15 and bla_CTX-M-3 were described in largely diffused plasmids of various Inc groups, including IncL/M and IncF, which were associated with the spread of several other ESBL genes, and the occurrence of these plasmids is linked to selection exerted by antimicrobial use.^7,29

Phylogenetic groups, ST and CC to which belonged the six typed CTX-M-producing E. coli were D/ST405 (CC405), A/ST10 (CC10), A/ST617 (CC10), and B1/ST1431. It has been shown that CTX-M type enzymes are strongly associated with phylogenetic group D.⁵ E. coli-D strains from various countries were found belonging to ST405, which was recently described with ST131 as E. coli clones, with pathogenic strains, spreading worldwide with CTX-M-15.⁴³ Phylogroups A and B1, to which belong commensal and environmental strains, are also involved in the carriage of CTX-M, including CTX-M-15. Genotype ST10/A was found harboring five different ESBLs (CTX-M-14, SHV-12, CTX-M-9, CTX-M-15, and CTX-M-32),³⁴ and CTX-M-15 was detected in ST167/A.⁴³

The 11 CTX-M-producing K. pneumoniae analyzed belonged to phylogenetic group KpI and to ST15, ST17, ST36, ST48, ST54, and ST147. The KpI cluster is dominantly represented by hospital and community isolates,¹³ and the most of these ST were described in several countries as carriers of CTX-M-15 enzyme, in Hungary (ST15 and ST147),¹¹ Spain (ST17 and ST36),³⁴ Tunisia (ST147),¹⁷ and south Korea (ST15, ST17 and ST48).²⁷

The study showed a multi-drug resistance in WWTP, more significant in final effluents, with resistance mechanisms including ESBLs production associated with PMQR determinants and mutations in topoisomerase genes. The presence of mobile elements in genetic environments, self-transferable broad host-range plasmids, and integrons portends a high potential for spread of resistance genes. In addition, phylogroups and ST of isolates are known as ubiquitous hosts of ESBL genes. These findings match in part with those described in clinical settings in Algeria and several other countries; they impose their consideration in clinical practice and a careful monitoring of effluents released into natural environments, especially when they are intended for a domestic and agricultural reuse.

Footnotes

Acknowledgments

This work was supported by grants from National Fund for the Research and National Research Programs of the Ministry of Higher Education and Scientific Research (Algeria). C. Torres is financially supported by the project SAF2012-35474 from the Ministerio de Economía y Competitividad of Spain and Feder. V. Estepa has a predoctoral fellowship from the University of La Rioja, Spain.

Disclosure Statement

No competing financial interests exist.

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