Antimicrobial resistant bacteria can become harboured in sediments of postindustrial estuaries. Subsequently, their resistance traits could be enriched by pollutants deposited in the sediments. Recent evidence strongly suggests this may pose hazards that not only affects the health care sector, but could also impact tourism and the aquaculture industries. The River Clyde, UK was chosen for this study due to its extensive industrial history, and three sites were chosen to sample from representing different levels and types of industrial activities—two highly polluted and one relatively “pristine” site

Henriquez, Fiona L.

Hursthouse, Andrew

Knapp, Charles

McLellan, Ian

Peshkur, Tatyana

Rodgers, Kiri

Tonner, Rebecca

Williams, Roderick

University of Strathclyde Institutional Repository

Tonner, Rebecca and Rodgers, Kiri and Peshkur, Tatyana and McLellan, Ian and Williams, Roderick and Hursthouse, Andrew and Henriquez, Fiona L. and Knapp, Charles (2018) Co-selection of antibiotic resistance in Gram-negative bacteria caused by pollution legacy in the Clyde estuary. In: Mineral Society of Great Britain & Ireland, 2018-06-14 - 2018-06-15, University of Strathclyde. (Unpublished) , This version is available at https://strathprints.strath.ac.uk/64735/Strathprints is  designed  to  allow  users  to  access  the  research  output  of  the  University  of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may  not  engage  in  further  distribution  of  the  material  for  any  profitmaking  activities  or  any commercial gain. You may freely distribute both the url (https://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge. Any correspondence concerning this service should be sent to the Strathprints administrator: strathprints@strath.ac.ukThe Strathprints institutional repository (https://strathprints.strath.ac.uk) is a digital archive of University of Strathclyde research outputs. It has been developed to disseminate open access research outputs, expose data about those outputs, and enable the management and persistent access to Strathclyde's intellectual output.RESEARCH POSTER PRESENTATI ON DESI GN © 2015www.PosterPresentations.comINTRODUCTION OBTAINING	 ISOLATESRIVER	CLYDE	UK	All	 19	River	 Clyde	 sites	 were	 rank	summed	 against	 Potentially	 Toxic	 Elements	concentrations	found	and	3	sites	 were	 chosen	for	susceptibility	 testing-2	polluted	and	 1	pristine.	37	isolates	 were	 tested	for	susceptibilities	 to	an	array	of 	PTEs	 and	antibiotics.SUSCEPTIBILITY	 TESTINGIn	order	to	determine	 the	correlation	 between	 the	potentially	 toxic	 elements	found	within	the	3	chosen	sites	 principal	 component	analysis	 was	used.	Where	 the	values	 lie	 between	 the	3	components	highlights	 the	 variation	between	 source	 of 	pollutant	and		how	the	MICs	 and	MBCs	 for	bacteria	 isolate	correlate.PCA	ANALYSIS BIIVARIATE	 ANALYSISFig.	 9 	Signif icant	 bivariate	 correlations	 between	MIC,	 MBC	and	 IC50	of 	toxicants	 in	terms	 of 	geochemical	 conditions.	 All	variables	 were	 log2	transformed	prior	to	analysis	 for	both	PCA	and	Bivariate	 analysis.	HIGH-THROUGHPUT	 GENE	 ARRAY	QPCR19	sites	 of 	the	River	 Clyde	 were	 chosen	 spanning	the	 estuary	 in	order	 to	obtain	a	 comprehensive	 overview	 of 	the	range	 of 	pollution	in	the	river.	(1)Department	of	Civil	and	Environmental	Engineering,	University	of	Strathclyde,	Glasgow,	(2) University	of	West	of	Scotland,	Paisley	UK (rebecca.tonner@strath.ac.uk)Rebecca	Tonner	(1),	Kiri	Rodgers	(2),	Tanya	Peshkur (1),	Ian	MacLellan	(2),	Roderick	Williams	(2),	Andrew	Hursthouse (2),	Fiona	Henriquez	(2),	Charles	Knapp	(1)	CO-SELECTION OF ANTIBIOTIC RESISTANCE IN GRAM-NEGATIVE BACTERIACAUSED BY POLLUTION LEGACY IN THE CLYDE ESTUARYAntimicrobial	 resistant	 bacteria	 can	 become	 harboured	in	 sediments	 of 	post-industrial	estuaries.	Subsequently,	 their	 resistance	 traits	could	be	 enriched	 by	 pollutants	deposited	in	the	 sediments.	 Recent	 evidence	 strongly	 suggests	 this	 may	 pose	hazards	that	not	only	affects	 the	health	 care	 sector,	but	could	also	 impact	 tourism	and	the	aquaculture	 industries.The	 River	 Clyde,	 UK	was	 chosen	for	this	study	due	to	its	extensive	 industrial	history,	and	three	 sites	 were	 chosen	 to	sample	 from	representing	 dif ferent	levels	 and	 types	 of 	industrial	activities—two	 highly	 polluted	and	one	 relatively	“pristine”	 site.	Higher	 concentrations	of 	metals	 in	 the	environment	 correlated	 to	antibiotic	 resistance	 and	higher	MICs	 to	metals	 than	 among	 bacteria	 found	in	less	 polluted	 sites.	 These	 results	 also	 provides	some	 correlation	 between	the	absolute	 levels	 of 	the	zinc	resistant	 gene	 ZntA found	in	the	sites	 via	quantitative	 PCR	for	the	gene.128.  64.  32.  16 8.    4 2 1 0.5. 0.25. 0.125 0.0625 0MICToxican t	 	 B io -Me tric Ge ochemical 	Co rrelationsCoppe r	 MICpH	 (r=.4 4 6**), 	Co	 (r=5 20 **), 	Cu	 (r=44 5**), 	Ni	 (.4 15*)IC5 0 pH	 (r=.4 4 6**), 	Co	 (r=5 10 **), 	Cu	 (r=52 0**), 	Ni	 (.4 50*)Nicke l	 MIC 	 Co 	 (r=.4 0 4*), 	Cu	 (.429 **), 	Ni 	(.35 9*)Co lbalt	MIC 	 Co 	 (r=.4 5 9**), 	Cu 	(.40 3*), 	Ni 	(.55 2**)IC5 0 Co 	 (r=-.3 3 2*)Chromium	(6) MIC 	 cr6 	 (r=	 -.3 3 9*)Pe n ici l l inMIC 	 As	 (r=-.3 3 0 *)IC5 0 As	 (r=-.3 8 8 *), 	Zn 	(r=-.38 7*)Trime thoprimMIC 	Zn 	 (r=.4 7 1 **)IC5 0 As	 (r=-.3 2 5 *), 	Cu	 (r=.3 70*), 	Zn 	(r=-.680 **)MBC Zn 	 (r=.5 0 5 **)Te tracyclineMIC 	pH	 (r=-.3 8 6	*), 	As	 (r=-.37 7*), 	Cu	 (r=-.3 28 *)IC5 0pH	 (r=.4 8 7**), 	As	(r=-.46 4**), 	Co 	(r=-.431 **), 	Cu	 (r=-.414 *),	Ni	 (-.3 3 1 *)MBC pH	 (r=-.3 6 7*)Co listin MBC Zn 	 (r=-.4 0 1 **)Doripe nem MBC As	 (r=-.3 4 6 *), 	Ni 	(r=-.363 *)r =	 Pear son	 cor r elat ion. 	 	 	 *s ig. 	 <0. 010	 	 **s ig. 	 <0. 005	 	 	 	 	 	 	 	 	 	 	 	Fig.1	 Core	 depths	were	 taken	 from	all	 sites	 using	 core	sampling	method	shown	above	 and	the	 cores	 divided	 into	10cm	segments	to	represent	 varying	 time	 periods	in	 the	soil.Fig.3	 Gram-negative	enteric	microorganisms	 were	obtained	 from	each	 of 	the	10cm	 core	depths.	 	Fig.2- Core	sample	 examplePaleo-limnology	 is	the	 examination	of 	past	environmental	 conditions	through	deposition	layers	 in	sedimentFig.4	Minimum	 Inhibitory	96-well	assay	 depicting	 the	resistance	 of 	isolated	 bacteria	 to	selected	 antibiotic.	 Values	 are	 in	 two-fold	dilution	and	units	are	 mg/L.Fig.	 8- PCA	analysis	 of 	the	MBC	 values	 in	Cardross,	Dumbarton	West	and	Clydebank,	 MIC	 and	MBC	values	Fig.	 7- PCA	analysis	 of 	the	MIC	 values	 in	Cardross,	 Dumbarton	West	 and	 ClydebankFig.	 6- PCA	analysis	 of 	the	PTE	 values	 in	Cardross,	 Dumbarton	West	 and	 ClydebankFigure	 5- MICs	for	bacteria	 isolated	 from	core	depths	from	the	3	sites:	 Cardross,	Dumbarton	West	 and	 Clydebank.v From	the	 results	 obtained	it	is	 clear	 that	gram-negative	 bacteria	 isolated	from	an	area	 with	an	extensive	 industrial		 pollution	history	show	higher	minimum	 inhibitory	concentrations	 and	minimum	 bactericidal	concentrations	to	a	 range	 of 	both	PTEs	 and	antibiotics.v In	general,	 genes	 for	resistance	 mechanisms	 were	 shown	to	be	highest	within	0-10cm	of 	soils	however	 when	examining	 data	 from	deeper	 cores,	isolated	 bacteria	 still	 harbour	resistance	 traits	 to	both	PTEs	and	antibiotics.v Through	a	 combination	of 	susceptibility	 assay	 data,	 qPCR	and	high	throughput	gene	 array	 qPCR	technology	 and,	it	is	 clear	 co-selection	 of 	PTEs	 and	 antibiotic	resistance	 does	 occur,	and	this	impacts	 bacteria	 that	are	 potential	 human	pathogens.	Fig.10	 Data	 shows	the	 presence	 of 	resistance	 gene	 prof iles	in	 core	depths	obtained	 from	each	 site.	All	 values	 represent	 genes/16S-rRNA	 (total	bacteria) 	 that	have	 been	 log-transformed	(e.g.,	 -1 	=	10%,	-2	=	 1%,	-3	=	0.1%	population	with	the	gene).CONCLUSIONSMINIMUM	 INHIBITORY	 CONCENTRATIONSApplied	 Biosciences	 gene-chip	 analysis	 was	carried	 out	on	the	3	sites	 chosen	for	the	susceptibility	 assays.	

Co-selection of antibiotic resistance in Gram-negative bacteria caused by pollution legacy in the Clyde estuary

Recent evidence indicate that numbers of antibiotic resistant bacteria within contaminated landscapes are significantly higher than that of uncontaminated land. The legacy of pollution and the inability to remove pollution by-products such as heavy metals can cause co-selection for antibiotic resistant genes within bacterial species. Three sites with different pollution levels were chosen to compare the levels of antibiotic/heavy metal resistance genes: Clydebank, Dumbarton West and Cardross. Using sediment cores, we were able to get a historical representation of pollution and resistant bacteria levels from the past to the presen

Tonner, Rebecca Lee

Henriquez, Fiona

Tonner, Rebecca Lee and Peshkur, Tatyana and Williams, Roderick and McLellan, Ian and Hursthouse, Andrew and Henriquez, Fiona and Knapp, Charles (2018) Co-selection of antibiotic resistance in Gram-negative bacteria caused by pollution legacy in the Clyde estuary. In: Strathwide 2018, 2018-06-06 - 2018-06-06, University of Strathclyde. (Unpublished) , This version is available at https://strathprints.strath.ac.uk/64737/Strathprints is  designed  to  allow  users  to  access  the  research  output  of  the  University  of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may  not  engage  in  further  distribution  of  the  material  for  any  profitmaking  activities  or  any commercial gain. You may freely distribute both the url (https://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge. Any correspondence concerning this service should be sent to the Strathprints administrator: strathprints@strath.ac.ukThe Strathprints institutional repository (https://strathprints.strath.ac.uk) is a digital archive of University of Strathclyde research outputs. It has been developed to disseminate open access research outputs, expose data about those outputs, and enable the management and persistent access to Strathclyde's intellectual output.INTRODUCTIONHYPOTHESIS (1)Department	of	Civil	and	Environmental	Engineering,	University	of	Strathclyde,	Glasgow,	(2) University	of	West	of	Scotland,	Paisley	UK (rebecca.tonner@strath.ac.uk)Rebecca	Tonner	(1),	Kiri	Rodgers	(2),	Tanya	Peshkur (1),	Ian	MacLellan	(2),	Roderick	Williams	(2),	Andrew	Hursthouse (2),	Fiona	Henriquez	(2),	Charles	Knapp	(1)	0. 0020. 0040. 0060. 0080. 00100. 00120. 000 - 1010	-2020 - 3030 - 40 40 - 490 - 1010	-2020 - 3030 - 40 40 - 5050 - 57.50 - 1010	-2020 - 3030 - 40 40 - 5050 - 57Zinc	Concentration	mg/kgCore 	de p ths	segments	 (cm)Zinc	levels 	in	core	depths 	of	Clydebankv From	the	results	obtained	it	is	clear	that	gram-negative	bacteria	isolated	from	an	area	with	an	extensive	industrial		pollution	history	show	higher	minimum	inhibitory	concentrations	and	minimum	bactericidal	concentrations	to	a	range	of	both	PTEs	and	antibiotics.v In	general,	genes	for	resistance	mechanisms	were	shown	to	be	highest	within	0-10cm	of	soils	however	when	examining	data	from	deeper	cores,	isolated	bacteria	still	harbour	resistance	traits	to	both	PTEs	and	antibiotics.v Through	a	combination	of	qPCR,	high	throughput	gene	array	qPCR	technology	and	susceptibility	assay	data,	it	is	clear	co-selection	of	PTEs	and	antibiotic	resistance	does	occur,	and	this	impacts	bacteria	that	are	potential	human	pathogens.	0. 0020. 0040. 0060. 0080. 00100. 00120. 000 - 10 10	-20	20	-280 - 10 10	-20	20	-260 - 10 10 - 18Zinc	Concentration	(mg/kg)Core 	de p ths	segments	 (cm)Zinc	levels 	in	core	depths 	of	cardrossSit e	 His t or y- Ver y	lit t le	 his t or y	of 	polut ion0. 0020. 0040. 0060. 0080. 000 - 1010	-2020 - 3030 - 40 0 - 1010	-2020-290 - 1010	-2020-3030 - 40 40 - 45Zinc	Concentration	mg/kg	Core 	de p ths	segments	 (cm)Zinc	levels 	in	core	depths 	of	Dumbarton	westSit e	 His t or y -low	 FeSewing	m achine	f act or y	0 2 4 6 8050100150Inhibition of Isolate from Clydebank core 40-50cmConcentration (mM)Percentage Inhibition (%)HIGH-THROUGHPUT GENE ARRAY QPCRApplied	 Biosciences 	gene-chip	 analys is 	was 	carried	out	 on	 the	3	s ites 	chosen	for	 the	 susceptibility	assays .	Data	 shows 	the	 presence	of	 res is tance	gene	 profiles 	in	 core	depths 	 obtained	 from	 each	s ite.	All	values 	represent	 genes/16S-rRNA	 (total	 bacteria)	 that	 have	been	 log-transformed	 (e.g.,	-1	 =	10%,	-2	 =	1%,	-3	=	0.1%	population	 with	the	 gene).IC 5 0 	– 0 .4mMR²=0 .9 7 6MINIMUM INHIBITORY  CONCENTRATION & qPCR RESULTS   Minimum	 Inhibitory	 Results 	were	compared	 to	 PTE	levels 	in	the	soils 	and	their	 depths 	 to	identify	 if	 a	correlation	 between	 the organism	 isolated	 and	the	 res is tance	that	 organism	 showed	to	 the	 individual	 PTE	upon	 re-exposure.	 An	 example	 of	the	 data	 has 	been	given	below.PCA ANALYSIS OF PTE VALUES IN CLYDE SITES Figu re	4 	– Con cen tration	of	Zinc	at	varyin g	co re	dep th s	at	C lydebank,	R iver	C lyde	UKFigu re	2 -IC5 0 	Cu rve	o f	C lydebank	Co re	0-10 cm- Calcu lating	 th e	p ercen tage	 in h ib i tion	 to 	Nickel 	Ch lo ride	p resen ted 	b y	 the	 i so late	 from	the	C lydebank	Si te.Fig.	1- PCA	analys is 	of	the	PTE	values	in	Cardross ,	Dumbarton	West	and	Clydebank96-WELL SUSCEPTIBILITY ASSAY & qPCR96-well assays were read sp ectro -pho tom etrically to dete rmine IC50 value. Mini mum Inhi bito ry Values w ere visualizedby us ing reazu rin as a n oxidiz ing reagen t an d Mi nimu m Bac tericidal Concen tra tions we re ob tained by pla ting the wellswhich showed no colour change (and subsequently no viability of bacteria).Figu re	3 -IC5 0 	Cu rve	o f	C lydebank	Co re	40 -5 0cm- Calculating	th e	p ercen tage	 in h ib i tion	 to	Nickel 	Chloride	presented 	by	 the	i so late	 from	the	C lydebank	Si teFigu re	3 - Con cen tration	of	Zinc	at	varyin g	co re	dep ths	at	Card ro ss, 	R iver	C lyde	UK.Figu re	5 - Con cen tration	of	Zinc	at	varyin g	co re	dep ths	at	Dumbarton 	West, 	R iver	C lyde	UK .051015202530MIC	Values	(µg/ml)Cor e	 dept h	 segm ent s 	 ( cm )Zinc	 re sistance 	 shown 	from	 Iso late d 	bacte ria	 in 	C lyde bankHigher	 MICs 	 in	isolated	 organisms 	from	 sediments 	with	 higher	 levels 	of	z inc	in	the	 sediments .	 These	results 	also	provides 	 some correlation	 between	 the	absolute	 levels 	of	the	 z inc	res is tant	gene	 ZntA found	 in	the	 s ites 	via	quantitative	 PCR	 for	 the	 gene.010203040506070DWB-S-1.0-10DWB-S-1.10-20DWB-S-1.20-30DBW-S-1.30-40DWB-S-2.0-10DWB-S-2.10-20DWB-S-2.20-29DWB-S-3.0-10DWB-S-3.10-20DWB-S-3.20-30DBW-S-3.30-40DBW-S-3.40-45MIC	Values	(µg/ml)Zinc	 re sistance 	 o f	 Iso late d 	Bacte ria	 from	Dumbarton 	 We st051015202530CAR-S-1.0-10CAR-S-1.10-20CAR-S-1.20-28CAR-S-2.0-10CAR-S-2.10-20CAR-S-2.20-26CAR-S-3.0-10CAR-S-3.10-18MIC	Values	(µg/ml)Cor e	 dept h	 segm ent s 	 ( cm ) 	Zinc	 re sistance 	 o f	 Iso late d 	Bacte ria	 from	Card rossFigu re	6 -M inimum	 Inhibi tory	Concen tration	of	Cardross	 Isolates	 to 	Zinc	Su l fate.Figu re	7 –M inimum	 Inhibi tory	Con centration 	o f	 Clydebank	 Isolates	 to	Zin c	Su l fate.Figu re	8 –M inimum	 Inhibi tory	Con centration 	o f	Dumbarton	West	 	Iso lates	 to 	Zin c	Su lfate.In	the	Clyde,	UK	co-selection	antibiotic	resistance	occurs	in	microorganisms,	promoted	by	stress	from	potentially	toxic	element	(PTE)	presence,	and	impacts	bacteria	that	could	possibly	be	human	pathogens.	Higher	concentrations	of	PTE	in	the	environment	correlates	to	higher	MIC	to	the	PTE,	and	ultimately	to	higher	antibiotic	resistance	levels.Tab le	2 - Heat	Map 	o f	an tib io tic	 resistan ce	genes	 in 	3	cho sen 	sites	o f	R iver	Clyde,	UK .	Ul tra	blue	=	 lowest	valu e	 fo r	th e	gene	clu ster	among	al l 	 si tes/dep th ,	Ultra	 red 	=	highest	value	 fo r	th e	gene	cluster.qPCRReal–time P CR was used to de tect the presence of metal res is tance genes f rom ext racted DNA. Thisallowed comparisons to be ma de betwee n metal gene abu ndance, the r es is tance genes found in highthroughput gene array qPCR and the Minimum-Inhibitory Concentrations obtained.Recent	evidence	indicate	that	numbers	of	antibiotic	resistant	bacteria	within	contaminated	landscapes	are	significantly	higher	than	that	of	uncontaminated	land.	The	legacy	of	pollution	and	the	inability	to	remove	pollution	by-products	such	as	heavy	metals	can	cause	co-selection	for	antibiotic resistant	genes	within	bacterial	species.		Three	sites	with	different	pollution	levels	were	chosen	to	compare	the	levels	of	antibiotic/heavy	metal	resistance	genes:Clydebank,	Dumbarton	West	and	Cardross.Using	sediment	cores,	we	were	able	to	get	a	historical	representation	of	pollution	and	resistant	bacteria	levels	from	the	past	to	the	present051 01 52 02 53 0CAR-S-1.0-10CAR-S-1.10-20CAR-S-1.20-28CAR-S-2.0-10CAR-S-2.10-20CAR-S-2.20-26CAR-S-3.0-10CAR-S-3.10-18Absolute	quantification	(genes/g-sediment)	x100000Le ve l	o f	Zn tA ge ne 	 in 	Card ross	Se d ime nts	Figu re	9 - The	abundan ce	of	Zn tAgene	 (copies	per	gram	of	 soi l	DNA)	quanti fied 	by	qPCR	 )	in	Card ross	Core	Depths.0 .0 01 0 .0 02 0 .0 03 0 .0 0DWB-S-1.0-10DWB-S-1.10-20DWB-S-1.20-30DBW-S-1.30-40DWB-S-2.0-10DWB-S-2.10-20DWB-S-2.20-29DWB-S-3.0-10DWB-S-3.10-20DWB-S-3.20-30DBW-S-3.30-40DBW-S-3.40-45Absolute	quantification	(genes/g-sediment)	x100000Core 	de p ths	 from	Dumbarton 	West	se gments	 (cm)Le ve l	o f	Zn tA	ge ne 	 in 	Dumbarton 	We st	Se d ime nts	Figu re	1 0 - The	abundan ce	of	ZntAgene	 (cop ies	per	gram	o f	 soi l 	DNA)	quan ti fied 	b y	qPCR	 )	 in	Clydebank	Co re	Dep ths.051 01 52 02 53 03 5CYV-S-1.0-10CYV-S-1.10-20CYV-S-1.20-30CYV-S-1.30-40CYV-S-1.40-49CYV-S-2.0-10CYV-S-2.10-20CYV-S-2.20-30CYV-S-2.30-40CYV-S-2.40-50CYV-S-2.50-57.5CYV-S-3.0-10CYV-S-3.10-20CYV-S-3.20-30CYV-S-3.30-40CYV-S-3.40-50CYV-S-3.50-57Absolute	quantification	og(genes/g-sediment)	x100000Core 	de p ths	 from	C lyde bank	samp le s	(cm)Le ve ls	o f	Zn tA ge ne 	 in 	C lyde bank	Samp le sFigu re	1 1 - The	abundan ce	of	ZntAgene	 (cop ies	per	gram	o f	 soi l 	DNA)	quan ti fied 	b y	qPCR	 )	 in	Dumbarton 	West	Core	Dep ths.IC 5 0 	– 1 .2 5mMR²=0 .9 5 7In	order	 to	determine	 the	 correlation	 between	 the	 potentially	 toxic	 elements 	 found	 within	 the	3	chosen	 s ites 	principal	 component	 analys is 	was 	used.	Where	the	 values 	lie	between	 the	 3	components 	 highlights 	 the	variation	 between	 source	 of	pollution.CONCLUSIONS

https://strathprints.strath.ac.uk/64735/1/Tonner_MinSoc_2018_Co_selection_of_antibiotic_resistance_in_Gram_negative_bacteria_caused_by_pollution.pdf

Co-selection of antibiotic resistance in Gram-negative bacteria caused by pollution legacy in the Clyde estuary

Abstract

Similar works

Full text

Available Versions

University of Strathclyde Institutional Repository

University of Strathclyde Institutional Repository