First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia

SAMAR BEN MILOUD; OLFA DZIRI; SANA FERJANI; MUNTASIR MD ALI; MOHAMED MYSARA; ILHEM BOUTIBA; ROB VAN HOUDT; CHEDLY CHOUCHANI

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First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia

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| Dec 06, 2021

Polish Journal of Microbiology

Volume 70 (2021): Issue 2 (June 2021)

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Cite

Published Online: Dec 06, 2021

Page range: 161 - 174

Received: Sep 20, 2020

Accepted: Feb 15, 2021

DOI: https://doi.org/10.33073/pjm-2021-012

Keywords
contaminated water, environmental bacteria, heavy metals (HM), antibiotics (AB), cross-resistance

© 2021 Samar Ben Miloud et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Phylogenetic tree based on the partial 16S rRNA gene sequences of the 39 isolates. Ten colors used to distinguish ten different sampling sites classified from north to south of Tunisia: Dark blue: Menzel Jemil; Orange: Iron factory; Red: Tinjahwedi Bizerte; Cyan: Collector between wastewater treatment plant (CWTP) of Beja; Green: Marsh Sejoumi; Yellow: Milian Rades Wedi; Light purple: Majerda River; Pink: Lebnawedi Cap Bon; Dark purple: Om Larayes Gafsa; Grey: Golf of Gabes.

Detection by PCR of heavy metal resistance genes.a – Amplicon of silE of Enterobacter cloacae 27 (400 bp);b – Amplicon of cusA of Klebsiella pneumoniae 13 (410 bp);c – Amplicon of merA of Pseudomonas putida 26 (285 bp);M – Size Marker 1 kb Plus.

Sequence alignment of the partial SilE protein from 39 isolates with SilE from pMG101 (SilE AAD11743). Letters shows res idues different from the consensus. Conserved histidine and methionine residues are marked above with either a circle or a square, respectively.

Sequence alignment of the partial Cation efflux system protein CusA from 10 isolates with E. coli P30854. Different residues from the consensus are showed by one letter. Conserved residues are represented by points.

Sequence alignment of the partial mercuric reductase protein MerA from 8 isolates with E. cloacae (MerA EU081910). Different residues from the consensus are showed by one letter. Conserved residues are represented by points.

Phenotypic and molecular characteristics of antibiotic and heavy metal resistant isolates collected from polluted water in Tunisia.

Strains	Sites	MICs of HM (µg/ml) Ag²⁺ Cu²⁺ Hg²⁺	HM resistance genes	AB resistance profile	AB resistance genes
Pseudomonas anguilliseptica 1	MJ. Bizerte	0.064 (R) 0.625(S) 0.08 (R)	silE, merA	AMP, ATM, FOS	bla_TEM
Alcaligenes eutrophus 2	MJ. Bizerte	0.064 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, CAZ	bla_TEM
Escherichia coli. 3	MJ. Bizerte	0.064 (R) 6 (R) 0.0025 (S)	silE, cusA	AMP, TIC, PIP, CXM, CFM, CAZ, ATM, GMN, NET, TOB, CTX	bla_TEM, blaCTX-M-1, qnrB
Staphylococcus aureus 4	MJ. Bizerte	0.064 (R) 3(R) 0.08 (R)	silE, cusA, merA	AMP, ATM, FOS, CIP, LEV	bla_TEM
Pseudomonas mendocina 5	IF of Bizerte MB	0.032 (R) 3(R) 0.08 (R)	silE, cusA, merA	AMP, ATM, FOS	bla_TEM
Alcaligenes eutrophus 6	IF of Bizerte MB	0.064 (R) 6(R) 0.005 (S)	silE, cusA	AMP, CAZ, SXT, CHL	bla_TEM
Klebsiella pneumoniae 7	IF of Bizerte MB	0.064 (R) 3(R) 0.08 (R)	silE, cusA, merA	AMP, TIC, FOX, FEP, ETP, AMC, CAZ, IMP, SXT, CTX, FOS, CLS, NOR, CIP, GMN, AKN, NET, TOB, NFE, MNO, TET	bla_TEM, bla_SHV, blaCTX-M-1, bla_OX48, qnrB
Pseudomonas putida 8	IF of Bizerte MB	0.064 (R) 1.5 (S) 0.005 (S)	silE	AMP, TIC, TCC, PIP, FEP, CAZ, ATM, FOS	bla_TEM
Alcaligenes faecalis 9	IF of Bizerte MB	0.064 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, CAZ	bla_TEM
Pseudomonas mendocina 10	Tinjah wedi, Bizerte	0.064 (R) 1.5 (S) 0.08 (R)	silE, cusA, merA	AMP, ATM, FOS	bla_TEM
Pseudomonas mendocina 11	Tinjah wedi, Bizerte	0.032 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, TCC, FOS	bla_TEM
Alcaligenes faecalis 12	Tinjah wedi, Bizerte	0.064 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, CAZ, CHL	bla_TEM
Klebsiella pneumoniae 13	Tinjah wedi, Bizerte	0.032 (R) 3 (R) 0.04 (R)	silE, cusA, merA	AMP, TIC, AMC, NAL, NOR, CHL, TGC, MNO, TET	bla_TEM, bla_SHV
Pseudomonas fluorescens 14	CWTP of Beja	0.064 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, TIC, ATM, FOS, IMP, MEM,	bla_TEM
Pseudomonas putida 15	CWTP of Beja	0.064 (R) 1.5 (S) 0.08 (R)	silE, merA	AMP, TIC, TCC	bla_TEM
Pseudomonas putida 16	CWTP of Beja	0.008 (S) 3 (R) 0.04 (R)	cusA, merA	AMP, TIC, TCC, PIP, FEP, CAZ, ATM, FOS	bla_TEM
Aeromonas salmonicida 17	Marsh Sejoumi	0.032 (R) 1.5 (S) 0.08 (R)	silE, merA	AMP, TIC	bla_TEM
Alcaligenes eutrophus 18	Marsh Sejoumi	0.008 (S) 0.625 (S) 0.08 (R)	merA	AMP, CAZ	bla_TEM
Pseudomonas alcaligenes 19	Marsh Sejoumi	0.0064(R) 3 (R) 0.08 (R)	silE, cusA, merA	AMP, TIC, PIP, TCC, FOS	bla_TEM
Enterobacter cloacae 20	Marsh Sejoumi	0.064 (R) 6 (R) 0.08 (R)	silE, cusA, merA	AMP, TIC, FOX, AMC, CTX	bla_TEM, bla_OXA-1, bla_SHV, blaCTX-M-9
Bacillus coagulans 21	Milian Rades Wedi	0.064 (R) 3 (R) 0.02 (R)	silE, cusA, merA	AMP, TIC, TCC, PIP, FEP, CAZ, ATM, FOS	bla_TEM
Alcaligenes eutrophus 22	Milian Rades Wedi	0.064 (R) 0.625 (S) 0.08 (R)	silE, merA	AMP, CAZ, SXT, CHL	bla_TEM
Pseudomonas putida 23	Milian Rades Wedi	0.004 (S) 0.625 (S) 0.005 (S)	–	AMP, ATM, FOS	bla_TEM, bla_SHV
Alcaligenes eutrophus 24	Majerda River	0.064 (R) 1.5 (S) 0.005 (S)	silE	AMP, CAZ	bla_TEM
Serratia marcescens 25	Majerda River	0.064 (R) 1.5 (S) 0.02 (R)	silE, merA	AMP, TIC, FOX, AMC	bla_TEM, bla_OXA-1, bla_SHV
Pseudomonas putida 26	Majerda River	0.032 (R) 0.75 (S) 0.02 (R)	silE, merA	AMP, FOS, ATM, LEV	bla_TEM
Enterobacter cloacae 27	Lebna wedi C.B	0.064 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, TIC, FOX, AMC, TGC, MNO, TET	bla_TEM, bla_OXA-1
Serratia fonticola 28	Lebna wedi C.B	0.064 (R) 1.5 (S) 0.005 (S)	silE	AMP, TIC, AMC, CTX, CLS	bla_TEM, blaCTX-M-9
Alcaligenes faecalis 29	Lebna wedi C.B	0.032 (R) 3 (R) 0.005 (S)	silE, cusA	AMP, CAZ	bla_TEM
Klebsiella pneumoniae 30	Lebna wedi C.B	0.064 (R) 6 (R) 0.005 (S)	silE, cusA	AMP, TIC, AMC	bla_TEM, bla_SHV
Pseudomonas fluorescens 31	Om Larayes, Gafsa	0.064 (R) 3 (R) 0.08 (R)	silE, cusA, merA	AMP, TIC, TCC, PIP, FEP, ATM, IMP, MEM, FOS	bla_TEM
Aeromonas salmonicida 32	Om Larayes, Gafsa	0.064 (R) 0.625(S) 0.005 (S)	silE	AMP	bla_TEM
Aeromonas salmonicida 33	Om Larayes, Gafsa	0.064 (R) 1.5 (S) 0.08 (R)	silE, merA	AMP, TIC, FEP, CAZ, ATM	bla_TEM
Pseudomonas putida 34	Om Larayes, Gafsa	0.032 (R) 1.5 (S) 0.08 (R)	silE, merA	AMP, TIC, TCC, PIP, TZP, CAZ, ATM	bla_TEM
Pseudomonas fluorescens 35	Om Larayes, Gafsa	0.0064 (R) 0.75 (S) 0.005 (S)	silE	AMP, TIC, TCC, ATM, MEM	bla_TEM
Pseudomonas putida 36	Gulf of Gabes	0.064 (R) 3 (R) 0.008 (S)	silE, cusA	AMP, TIC, TCC, PIP, TZP, ATM, MEM	bla_TEM
Serratia marcescens 37	Gulf of Gabes	0.064 (R) 1.5 (S) 0.005 (S)	silE	AMP, FOX, AMC, TGC, MNO, TET	bla_TEM
Pseudomonas fluorescens 38	Gulf of Gabes	0.0064 (R) 6 (R) 0.08 (R)	silE, cusA, merA	AMP, TIC, TCC, PIP, TZP, FEP, CAZ, ATM, MEM, LEV, FOS	bla_TEM
Klebsiella pneumoniae 39	Gulf of Gabes	0.032 (R) 3 (R) 0.0025 (S)	silE, cusA	AMP, TIC, TCC, PIP, CFN, CXM, CFM, CAZ, FEP, ATM, GMN, NET, TOB	bla_SHV, blaCTX-M-1, qnrB

Primers, expected fragment size, and conditions of PCR experiments used for quinolones resistance encoding genes.

Multiplex	Target	Sequence of primer (5′–3′)	Size (bp)	Amplification conditions
3	qnrA	qnrA_FCAGCAAGAGGATTTCTCACG	630	95°C 15 min 94°C 30 sec 63°C 40 sec 30 cycles 72°C 90 sec 72°C 10 min
	qnrA	qnrA_RAATCCGGCAGCACTATTACTC	630
	qnrB	qnrB_FGGCTGTCAGTTCTATGATCG	488
	qnrB	qnrB_RGAGCAACGATGCCTGGTAG	488
	qnrC	qnrC_FGCAGAATTCAGGGGTGTGAT	118
	qnrC	qnrC_RAACTGCTCCAAAAGCTGCTC	118
	qnrD	qnrD_FCGAGATCAATTTACGGGGAATA	581
	qnrD	qnrD_RAACAAGCTGAAGCGCCTG	581
	qnrS	qnrS_FGCAAGTTCATTGAACAGGGT	428
	qnrS	qnrS_RTCTAAACCGTCGAGTTCGGCG	428

Sampling sites characteristics, locations, and their corresponding geographic coordinates.

Sites/numeration	Geographic coordinates	Location	Characteristics
Menzel Jemil, Bizerte: Site I	37°14′19″N,9°54′59″E	Industrial area	Waste and contamination from the textile industry and wiring throwing inside the Bizerte lagoon
Menzel Bourguiba, Bizerte: Site II	37°09′N, 9°47′E	Unit manufacturing printed circuits. In the Iron factory	Contamination by HM from the iron factory in the Bizerte lagoon. Urban and agricultural pollution
Tinjah wedi, Bizerte: Site III	37°10′N, 9°45′E	Near the lagoon of Bizerte	Agricultural pollution and compost contamination.
Beja: Site IV	36°43′30″N,9°10′55″E	Southwest of the city of Tunis Near the CWTP*	Urban and industrial area, the most known are wastewater and yeast factory
Essijoumi Lagoon: Site V	36°45′52″N,10°08′49″E	Contribution in the Gulf of Tunis	Lagoon receiving contamination from wastewater contamination and wastes from the capital Tunis.
Rades Milian River: Site VI	36°46′N,10°17′E	Industrial zone of Rades	High load alluvial estimated at 25 grams per liter. Receiving wastewater from two towns Rades and Ezzahra.
Majerda River: Site VII	37°7′0″N,10°13′0″E	A peninsula in far north-eastern Tunisia	Used for irrigation of the region’s agriculture
Lebna River: Site VIII	36°45′N,10°54′E	Inlet manifold sewage treatment plant	Agricultural coastal Plans can be found in the area of Cap Bon
Om Larayes, Gafsa: Site IX	34°28′59″N,8°16′01″E	The industrial platforms of phosphgyps activity	One of the known mining towns in Gafsa
Gulf of Gabes: Site X	34°05′37″N,10°26′13″E	The junction between the Eastern and Central Basin	Known by industry for the transformation of merchantable phosphate into Phosphoric Acid (H₃PO₄) and Chemical Fertilizers

Primers, expected fragment size and conditions of PCR experiments used for β-lactams resistance encoding genes.

Multiplex	Target	Primers sequences (5′–3′)	Size (pb)	Concentration (pmol/µl)	Volume (µl)	Amplification conditions
1	TEM	MultiTSO-T_F CATTTCCGTGTCGCCCTTATTC	800	0.4	0.4	94°C 10 min 94°C 40 sec 60°C 40 sec 30 cycles 72°C 1 min 72°C 7 min
	TEM	MultiTSO-T_R CGTTCATCCATAGTTGCCTGAC	800	0.4	0.4
	SHV	MultiTSO-S_F AGCCGCTTGAGCAAATTAAAC	713	0.4	0.4
	SHV	MultiTSO-S_R ATCCCGCAGATAAATCACCAC	713	0.4	0.4
	OXA-1-like	MultiTSO-O_F GGCACCAGATTCAACTTTCAAG	564	0.4	0.4
	OXA-1-like	MultiTSO-O_R GACCCCAAGTTTCCTGTAAGTG	564	0.4	0.4
2	CTX-M group 1	MultiCTXMGp1_F TTAGGAARTGTGCCGCTGTA	688	0.4	0.4
	CTX-M group 1	MultiCTXMGp1_R CGATATCGTTGGTGGTCCCAT	688	0.2	0.2
	CTX-M group 2	MultiCTXMGp2_F CGTTAACGGCACGATGAC	404	0.2	0.2
	CTX-M group 2	MultiCTXMGp1_R CGATATCGTTGGTGGTTCCAT	404	0.2	0.2
	CTX-M group 9	MultiCTXMGp9_F TCAAGCCTGCCGATCTGGT	561	0.4	0.4
	CTX-M group 9	MultiCTXMGp9_R TGATTCTCGCCGCTGAAG	561	0.4	0.4
	CTX-M group 8	CTX-Mg8/25_F AACTCCCAGACGCTCTAC	326	0.4	0.4
	CTX-M group 8	CTX-Mg8/25_R TCGAGCCGGAASGTGTAAT	326	0.4	0.4

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First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia

Article Category: original-paper

Published Online: Dec 06, 2021

Page range: 161 - 174

Received: Sep 20, 2020

Accepted: Feb 15, 2021

DOI: https://doi.org/10.33073/pjm-2021-012

Keywords
contaminated water, environmental bacteria, heavy metals (HM), antibiotics (AB), cross-resistance

© 2021 Samar Ben Miloud et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Phenotypic and molecular characteristics of antibiotic and heavy metal resistant isolates collected from polluted water in Tunisia.

Primers, expected fragment size, and conditions of PCR experiments used for quinolones resistance encoding genes.

Sampling sites characteristics, locations, and their corresponding geographic coordinates.

Primers, expected fragment size and conditions of PCR experiments used for β-lactams resistance encoding genes.

First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia

Article Category: original-paper

Published Online: Dec 06, 2021

Page range: 161 - 174

Received: Sep 20, 2020

Accepted: Feb 15, 2021

DOI: https://doi.org/10.33073/pjm-2021-012

Keywordscontaminated water, environmental bacteria, heavy metals (HM), antibiotics (AB), cross-resistance

© 2021 Samar Ben Miloud et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Phenotypic and molecular characteristics of antibiotic and heavy metal resistant isolates collected from polluted water in Tunisia.

Primers, expected fragment size, and conditions of PCR experiments used for quinolones resistance encoding genes.

Sampling sites characteristics, locations, and their corresponding geographic coordinates.

Primers, expected fragment size and conditions of PCR experiments used for β-lactams resistance encoding genes.

Keywords
contaminated water, environmental bacteria, heavy metals (HM), antibiotics (AB), cross-resistance