Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana.

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiellapneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

1. Introduction

The microbiology of chronic infected wounds, also on a molecular level, is poorly understood in sub-Saharan Africa (SSA) [1]. However, studies highlight the importance of antibiotic resistant Gram-negative bacteria [2,3,4,5,6].

From other parts in the world, in particular from industrialized countries, information on the microbiology and the role of biofilm-forming microorganisms causing such infections are well established [7,8,9,10].

In chronic wounds, Pseudomonas aeruginosa is amongst the most frequently isolated Gram-negative bacteria, associated with biofilm formation [11,12]. Tightly adhering biofilms pose a challenge in the diagnosis of P. aeruginosa using standard culturing methods [13].

In comparison, the role of Enterobacterales in chronic wounds has been much less characterized [14,15,16,17]. Studies have shown that geography seems to play a role in the estimation of their etiological relevance [18]. It was shown that skin colonization with Gram-negative bacteria is frequent in resource-limited (sub)tropical settings [19,20,21], in contrast to skin colonization of individuals from industrialized countries, where Gram-positive bacteria dominate [19]. Temperature and moisture have been discussed as likely reasons for the difference seen [22].

Isolation of potentially pathogenic bacteria from non-sterile sites like wounds does not necessarily indicate clinical relevance, which poses challenge to clinical interpretation.

In a recent study that focused on the overall bacterial composition of chronic wound infections in Ghana, from which the isolates for the present molecular analysis were taken, Enterobacterales and Pseudomonas aeruginosa constituted the majority of isolated bacterial strains [23]. A moderate proportion of ESBL-positive Enterobacterales suggests lower frequencies of antibiotic resistance [23] than what was recorded from other Ghanaian hospitals [5,24].

Within this study, we aim at characterizing clonal lineages, resistance-associated genetic elements and virulence genes of P. aeruginosa, the Klebsiella pneumoniae complex and Escherichia coli, which were recently isolated from chronic wounds of Ghanaian adult patients [23]. The molecular epidemiology of dominating clonal lineages and associated resistance genes will be assessed. Further, analysis of highly abundant virulence factors will be conducted.

2. Results

2.1. Clustering Based on Core Genome Multilocus Sequence Typing (cgMLST) Results

Of the 49 P. aeruginosa analyzed, a total of nine clusters comprising isolates without any recorded differences (n = 2) or with one or two alleles difference (n = 7) were found, suggesting closely related phylogeny (Figure 1). In addition to the clusters, 31 singletons with differences ranging from 80 to 3584 alleles were observed. MLST sequence types (ST) are indicated in Figure 1 and Table A1 and Table A2. Cluster sequence types included the following: ST244, ST245, ST381, ST554, ST856, ST1485, ST2033, ST3227 and ST3590.

Figure 1

Minimum spanning tree of P. aeruginosa based on 3867 targets (core genome). Isolate numbers are found within the nodes, and numbers between nodes indicate the number of different alleles. Isolates within clusters are colored based on MLST sequence type (ST). The ST types of white nodes are indicated in Table A1.

Table A1

Analysis of antimicrobial resistance determinants, ordered by strain and MLST type, of the assessed P. aeruginosa isolates. ST = Sequence type.

Sample ID	ST-Type	Acquired Resistance Determinants Against
		Beta Lacatams	Sulfonamids	Fosfomycin	Trimethoprim	Makrolides	Tetracyclinws	Fluoroquinolones	Chloramphenicol	Rifampicin	Aminoglycosides	Efflux Pumps	Amino Acid Exchanges Due to Point Mutations	Disinfectant Resistance Genes
017	ST 381			fosA					catB7		aph(3′)-IIb
022	ST 2483			fosA					catB7		aph(3′)-IIb
032	ST 3587		sul1	fosA	dfrA15		tet(G)		catB7		aph(3′)-IIb
069	ST 360			fosA				crpP-like	catB7		aph(3′)-IIb
081	ST 244			fosA					catB7		aph(3′)-IIb
082	ST 514			fosA					catB7		aph(3′)-IIb
088	ST 1682			fosA					catB7		aph(3′)-IIb
099	ST 244			fosA					catB7		aph(3′)-IIb
106	ST 1521			fosA					catB7		aph(3′)-IIb
114	ST 244			fosA				crpP-like	catB7		aph(3′)-IIb-like
137	ST 3014			fosA				crpP-like	catB7		aph(3′)-IIb-like
144	ST 245			fosA				crpP-like	catB7		aph(3′)-IIb
147	ST 245			fosA				crpP-like	catB7		aph(3′)-IIb
149	ST 381			fosA				crpP-like	catB7		aph(3′)-IIb
153	ST 704			fosA-like				crpP-like	catB7-like		aph(3′)-IIb- like
154	ST 244	,		fosA				crpP-like	catB7		aph(3′)-IIb-like
157	ST 2616			fosA					catB7-like		aph(3′)-IIb
160	ST 170			fosA-like							aph(3′)-IIb
162	ST 274			fosA				crpP-like	catB7		aph(3′)-IIb
180	ST 856			fosA					catB7		aph(3′)-IIb
183	ST 244			fosA					catB7		aph(3′)-IIb-like
186	ST 3588			fosA-like					catB7-like		aph(3′)-IIb-like
190	ST 871			fosA					catB7-like		aph(3′)-IIb
195	ST 988			fosA				crpP-like	catB7-like		aph(3′)-IIb-like
196	ST 2475			fosA				crpP-like	catB7		aph(3′)-IIb
198	ST 2476			fosA				crpP-like	catB7		aph(3′)-IIb
204	ST 639			fosA				crpP	catB7		aph(3′)-IIb-like
208	ST 132			fosA				crpP-like	catB7		aph(3′)-IIb
218	ST 856			fosA					catB7		aph(3′)-IIb
229	ST 270			fosA				crpP-like	catB7		aph(3′)-IIb
233	ST 3227			fosA					catB7		aph(3′)-IIb
236	ST 266			fosA					catB7		aph(3′)-IIb
238	ST 3589			fosA-like				crpP-like	catB7-like		aph(3′)-IIb-like
242	ST 3590			fosA-like
243	ST 3590			fosA-like					catB7-like		aph(3′)-IIb-like
272	ST 2033			fosA					catB7-like		aph(3′)-IIb-like
274	ST 2033			fosA					catB7-like		aph(3′)-IIb
278	ST 988			fosA				crpP-like	catB7-like
282	ST 554			fosA				crpP-like	catB7		aph(3′)-IIb
285	ST 554			fosA					catB7		aph(3′)-IIb
289	ST 1485			fosA					catB7		aph(3′)-IIb
290	ST 1485			fosA					catB7		aph(3′)-IIb
296	ST 235	blaTEM-1B, blaSCO-1	sul1	fosA			tet(G)		catB7-like		aph(3′)-IIb-like, aac(3)-IIa
298	ST 3227			fosA					catB7		aph(3′)-IIb
301	ST 3593			fosA-like					catB7-like		aph(3′)-IIb-like
302	ST 1755			fosA					catB7		aph(3′)-IIb
309	ST 3592			fosA like				crpP-like	catB7-like		aph(3′)-IIb-like
310	ST 532		sul1	fosA					catB7-like		aph(3″)-Ib, aph(6)-Id, aph(3′)-IIb
312	ST 381			fosA					catB7		aph(3′)-IIb

Acquired resistance genes for macrolides, rifampicin, resistance-associated point mutations, genes for efflux pumps or genes mediating tolerance against disinfectants were not detected.

Table A2

Analysis of antimicrobial resistance determinants, ordered by strain and MLST type, of the assessed K. pneumoniae isolates. ST = Sequence type.

Sample ID	ST-Type				Acquired Resistance Determinants Against
		Beta Lacatams	Sulfonamids	Fosfomycin	Trimethoprim	Macrolides	Tetracyclines	Fluoroquinolones	Chloramphenicol	Rifampicin	Aminoglycosides	Efflux Pumps	Amino Acid Exchanges Due to Point Mutations	Disinfectant Resistance Genes *
044	ST 327			fosA				oqxB, oqxA					ompK37 p.I70M, ompK37 p.I128M,ompK37 p.I128M, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK36 p.T254S, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxB, oqxA
060	ST 5379	bla TEM-1C	sul1, sul2	fosA	dfrA12	mph(A)		oqxA, oqxB, qnrS1	catA2-like		aph(6)-Id, aph(3″)-Ib, aph(3′)-Ia, aadA2, aac(3)-IIa		acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.T184P, ompK37 p.I70M, ompK37 p.I128M	oqxA, qacE, oqxB
073	ST 39	blaTEM-1B , bla CTX-M-15	sul1,	fosA	dfrA27	erm(B), mph(A)	tet(D)	oqxB, oqxA, aac(6′)-Ib-cr, qnrB2, aac(6′)-Ib-cr	catA2-like	ARR-3	aac(6′)-Ib-cr, aadA16, aac(3)-IIa, aac(6′)-Ib-cr, aph(3″)-Ib, aph(6)-Id		acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK37 p.I70M, ompK37 p.I128M, ompK37 p.N230G, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK36 p.T254S	oqxB, oqxA, qacE
100	ST 152	blaCTX-M-15 , bla OXA-1 , bla TEM-1B	sul2, sul1	fosA	dfrA1, dfrA27	mph(A)	tet(D)	aac(6′)-Ib-cr, oqxB, qnrB6, oqxA, aac(6′)-Ib-cr	catB3, catA1, catB3	ARR-3	aac(3)-IIa, aph(6)-Id, aph(3″)-Ib, aadA1, aadA16, aph(3′)-Ia, aac(6′)-Ib-cr, aac(6′)-Ib-cr		ompK36 p.N49S, ompK36 p.L59V, ompK36 p.G189T, ompK36 p.F198Y, ompK36 p.F207Y, ompK36 p.A217S, ompK36 p.T222L, ompK36 p.D223G, ompK36 p.E232R, ompK36 p.N304E, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK37 p.I70M, ompK37 p.I128M, ompK37 p.N230G	oqxB, oqxA
102	ST 514			fosA			tet(C)	oqxB, oqxA	catA1				ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK36 p.T254S, ompK37 p.I70M, ompK37 p.I128M, ompK37 p.N230G, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxB, oqxA
124	ST 399			fosA				oqxA, oqxB	catA1				ompK36 p.N49S, ompK36 p.L59V, ompK36 p.G189T, ompK36 p.F198Y, ompK36 p.F207Y, ompK36 p.A217S, ompK36 p.T222L, ompK36 p.D223G, ompK36 p.E232R, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.F197I, acrR p.K201M, ompK37 p.I70M, ompK37 p.I128M	oqxA, oqxB
146	ST 4		sul2	fosA			tet(D)	oqxA, oqxB	catA2-like					oqxA, oqxB
177	ST 17		sul1, sul2	fosA	dfrA15		tet(A)	oqxA, oqxB-like	catA1		aadA1, aph(3″)-Ib, aph(6)-Id		ompK37 p.I70M, ompK37 p.I128M, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK36 p.N49S, ompK36 p.L59V,ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232RompK36 p.T254S	qacE, oqxB-like, oqxA
181	ST 5380			fosA				oqxA, oqxB					ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK36 p.T254S,ompK37 p.I70M, ompK37 p.I128M, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxA, oqxB
184	ST 5381			fosA				oqxA-like, oqxB-like					ompK37 p.I70M, ompK37 p.I128M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191Q, ompK36 p.F198Y,ompK36 p.A217S, ompK36 p.N218H, ompK36 p.Q227N, ompK36 p.L229V, ompK36 p.N304E, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxA-like, oqxB-like
199	ST 17	blaCTX-M-15 , bla TEM-1B	sul2, sul1	fosA-like	dfrA16			oqxA, oqxB			aadA2b, aac(3)-IIa		acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK37 p.I70M, ompK37 p.I128M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.T86V, ompK36 p.S89T, ompK36 p.D91K, ompK36 p.A93S, ompK36 p.L191Q, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.Q227N, ompK36 p.L229V, ompK36 p.E232R,ompK36 p.H235D, ompK36 p.T254S	oqxA, oqxB, qacE
214	ST 6		sul1	fosA-like	dfrA14			oqxB-like, oqxA	catA1		aph(3′)-Ia		ompK37 p.I70M, ompK37 p.I128M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.G189T, ompK36 p.F198Y, ompK36 p.F207Y, ompK36 p.A217S, ompK36 p.T222L, ompK36 p.D223G, ompK36 p.E232R, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxB- like, oqxA
217	ST 3154	blaSCO-1 , bla TEM-1B	sul1, sul2	fosA	dfrA12, dfrA14		tet(A)	oqxA, oqxB-like	catA2-like		aph(6)-Id, aph(3″)-Ib, aac(3)-IIa, aadA2		acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK37 p.I70M, ompK37 p.I128M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.G189T, ompK36 p.F198Y, ompK36 p.F207Y, ompK36 p.A217S, ompK36 p.T222L, ompK36 p.D223G, ompK36 p.E232R, ompK36 p.N304E	oqxA, qacE, oqxB-like
220	ST 5382			fosA-like				oqxB-like, oqxA-like	catA1				ompK37 p.I70M, ompK37 p.I128M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191Q, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.Q227N, ompK36 p.L229V, ompK36 p.N304E, acrR p.P161R, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxB-like, oqxA-like
234	ST 109			fosA				oqxA, oqxB-like					ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK36 p.T254S, acrR p.P161R, acrR p.G164A, acrR p.F172S,acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK37 p.I70M, ompK37 p.I128M	oqxA, oqxB-like
240	ST 5383			fosA-like			tet(D)	oqxA, oqxB-like					ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK37 p.I70M, ompK37 p.I128M, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I,acrR p.K201M	oqxA, oqxB-like
248	ST 5384			fosA-like			tet(A)	oqxB-like, oqxA-like	catA1				ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F198Y, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK37 p.I70M, ompK37 p.I128M, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxB-like, oqxA-like
252	ST 607	bla TEM-1B	sul2, sul1	fosA-like	dfrA7		tet(A)	oqxB-like, oqxA	catA1		aph(3″)-Ib, aph(6)-Id		ompK37 p.I70M, ompK37 p.I128M, ompK37 p.N230G, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R, ompK36 p.T254S, acrR p.P161R,acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxB-like, oqxA, qacE
267	ST 36	blaCTX-M-15 , bla TEM-1B	sul2, sul1	fosA	dfrA27		tet(D)	aac(6′)-Ib-cr, oqxA, oqxB	catA2-like	ARR-3	aph(6)-Id, aph(3″)-Ib, aac(6′)-Ib-cr, aadA16, aac(3)-IIa, aph(6)-Id		ompK36 p.N49S, ompK36 p.L59V, ompK36 p.T184P, ompK37 p.I70M, ompK37 p.I128M, ompK37 p.N230G, acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M	oqxA, qacE, oqxB
277	ST 530	bla TEM-35	sul2	fosA-like	dfrA14		tet(D)	oqxA, oqxB-like			aph(3″)-Ib, aph(6)-Id		acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.L191S, ompK36 p.F207W, ompK36 p.A217S, ompK36 p.N218H, ompK36 p.D224E, ompK36 p.L228V, ompK36 p.E232R,ompK36 p.T254S, ompK37 p.I70M, ompK37 p.I128M	oqxA, oqxB-like
279	ST 5385			fosA				oqxA, oqxB					acrR p.P161R, acrR p.G164A, acrR p.F172S, acrR p.R173G, acrR p.L195V, acrR p.F197I, acrR p.K201M, ompK36 p.N49S, ompK36 p.L59V, ompK36 p.T184P, ompK37 p.I70M, ompK37 p.I128M	oqxA, oqxB

* qacE = quaternary ammonium compounds resistance and oqxB and oqxA = efflux pumps mediating resistance against disinfectants.

No clusters were identified among the 21 assessed K. pneumonia complex members, which were all singletons with differences ranging from 647 to 2244 alleles. K. pneumoniae complex sequence types are summarized in Figure 2. From the 12 E. coli isolates, three isolates in a cluster of close phylogenetic relationship were found (1× no allelic differences, 1 × 1 allele difference) (Figure 3). In addition to the cluster observed, nine singletons with differences ranging from 41 to 2365 alleles were recorded. The sequence type of the cluster was ST132 (Pasteur MLST scheme). Sequence types of all E. coli isolates are illustrated in Figure 3.

Figure 2

Minimum spanning tree of K. pneumoniae complex based on 2358 targets (core genome). Isolate numbers are found within the nodes, and the numbers between the nodes indicate the number of different alleles. Colors demonstrate the MLST sequence type of the isolates.

Figure 3

Minimum spanning tree of E. coli based on 2513 targets (core genome). Isolate numbers are found within the nodes, and the numbers between the nodes indicate the number of different alleles. Colors demonstrate the Pasteur sequence type of the isolates.

2.2. Identified Molecular Resistance Mechanisms in Correlation to Previous Phenotypic Antibiotic Resistance

Table 1 summarizes acquired antimicrobial resistance determinants for E. coli and acquired genes mediating tolerance to disinfectants. Data for P. aeruginosa and K. pneumoniae are presented in Table A1 and Table A2. Table A3, Table A4, Table A5, Table A6, Table A7 and Table A8 summarize the phenotypic resistance results as previously recorded [23].

Table 1

Analysis of antimicrobial resistance determinants, ordered by strain and MLST type, of the assessed E. coli isolates. ST = Sequence type.

Sample ID	ST-Type	Acquired Resistance Determinants Against
		Beta lacatams	Sulfonamids	Trimethoprim	Makrodlids	Tetracyclins	Fluoroquinolones	Chloramphenicol	Aminoglycosides	Efflux pumps	Amino acid exchanges due to point mutations	Disinfectant resistance genes *
041	ST 2	blaOXA-1, blaTEM-1B, blaCTX-M-15	sul1	dfrA17	mph(A)	tet(B)	aac(6′)-Ib-cr, aac(6′)-Ib-cr	catB3, catA1	aac(3)-IId, aac(6′)-Ib-cr, aadA5, aac(6′)-Ib-cr	mdf(A)	parE p.S458A, gyrA p.S83L, gyrA p.D87N, parC p.S80I	sitABCD, qacE
049	ST 3	bla TEM-1B	sul2, sul1,	dfrA12	mph(A)	tet(A)			aadA2, aph(3″)-Ib, aph(6)-Id	mdf(A)		sitABCD-like, qacE
068	ST 632	bla TEM-1B	sul3	dfrA12		tet(A)		cmlA1	aadA1, aadA2	mdf(A)-like	parE p.S458A, gyrA p.S83L, gyrA p.D87N, parC p.S80I
117	ST 4	bla TEM-1B	sul1, sul2	dfrA7		tet(A)		catA1	aph(6)-Id, aph(3 ″)-Ib	mdf(A)-like		sitABCD-like, qacE
152	ST 22	blaCARB-2, blaTEM-1B	sul1	dfrA1	ere(B)	tet(B)		catA1	aadA1, aadA2b	mdf(A)-like	gyrA p.S83L	qacE, sitABCD
176	ST 132	bla TEM-1B	sul1	dfrA7		tet(A)		catA1	aph(3 ″)-Ib, aph(6)-Id	mdf(A)		qacE, sitABCD
221	ST 132	bla TEM-1B	sul1	dfrA7		tet(A)		catA1	aph(6)-Id, aph(3 ″)-Ib	mdf(A)		qacE, sitABCD
222	ST 132	bla TEM-1B	sul1, sul2	dfrA7		tet(A)		catA1	aph(3 ″)-Ib, aph(6)-Id	mdf(A)		qacE, sitABCD
225	ST 506	blaTEM-1D, blaCTX-M-15	sul1, sul2	dfrA17	mph(A)	tet(A)		catA1	aadA5, aph(6)-Id, aph(3″)-Ib	mdf(A)-like	gyrA p.S83L, parE p.I529L	sitABCD-like, qacE
245	ST 2	bla TEM-1B	sul1	dfrA12	mph(A)	tet(B)	qepA4 (neu)	catA1	aadA2, aac(3)-IId	mdf(A)	parE p.S458A, gyrA p.S83L, gyrA p.D87N, parC p.S80I	qacE
270	ST 2	bla CTX-M-15				tet(B)		catA1		mdf(A)	gyrA p.S83L, gyrA p.D87N, parE p.S458A, parC p.S80I
299	ST 1018	bla TEM-1B	sul3	dfrA14		tet(A)	qnrS1			mdf(A)

* sitABCD = peroxides resistance, qacE = quaternary ammonium compounds resistance.

Table A3

Phenotypic resistance the P. aeruginosa strains. Data are missing for strains 198, 218 and 312, due to loss during subcultivation. MIC = minimum inhibitory concentration. N.a. = value missing due to loss of strain or failed reaction.

Sample ID	Piperacillin		Piperacillin/Tazobactam		Ceftrazidime		Cefepime		Imipenem		Meropenem		Gentamicin
	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation
17	≤4	S	≤4	S	≤1	S	≤1	S	≤0.25	S	≤0.25	S	≤1	S
22	≤4	S	≤4	S	≤1	S	≤1	S	≤0.25	S	≤0.25	S	≤1	S
32	≤4	S	8	S	2	S	2	S	1	S	≤0.25	S	≤1	S
69	≤4	S	≤4	S	4	S	2	S	1	S	≤0.25	S	≤1	S
81	≤4	S	8	S	2	S	≤1	S	1	S	1	S	≤1	S
82	16	S	8	S	4	S	2	S	2	S	≤0.25	S	≤1	S
88	≥128	R	≥128	R	≥64	R	32	R	≥16	R	4	I	≤1	S
99	8	S	8	S	4	S	2	S	2	S	1	S	≤1	S
106	≤4	S	8	S	2	S	2	S	2	S	1	S	≤1	S
114	≤4	S	8	S	2	S	≤1	S	2	S	1	S	≤1	S
137	16	S	8	S	4	S	2	S	2	S	2	S	≤1	S
144	16	S	8	S	4	S	2	S	2	S	1	S	≤1	S
147	8	S	≤4	S	4	S	8	S	2	S	0.5	S	4	S
149	8	S	8	S	4	S	2	S	≤0.25	S	≤0.25	S	≤1	S
153	≤4	S	8	S	2	S	≤1	S	1	S	≤0.25	S	≤1	S
154	64	R	≤4	S	≤1	S	≤1	S	2	S	0.5	S	≤1	S
157	16	S	8	S	4	S	4	S	2	S	≤0.25	S	2	S
160	≥128	R	32	R	16	R	32	R	8	I	8	I	8	R
162	64	R	32	R	8	S	8	S	2	S	1	S	2	S
180	16	S	8	S	4	S	2	S	2	S	≤0.25	S	≤1	S
183	8	S	8	S	4	S	2	S	2	S	0.5	S	≤1	S
186	16	n.a.	n.a.	S	4	S	2	S	2	S	≤0.25	S	≤1	S
190	16	S	8	S	4	S	2	S	2	S	0.5	S	≤1	S
195	8	S	8	S	4	S	≤1	S	2	S	≤0.25	S	≤1	S
196	≤4	S	≤4	S	2	S	≤1	S	2	S	0.5	S	≤1	S
198	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
204	8	S	8	S	2	S	≤1	S	2	S	≤0.25	S	≤1	S
208	8	S	8	S	4	S	2	S	2	S	≤0.25	S	≤1	S
218	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
229	8	S	8	S	4	S	2	S	1	S	≤0.25	S	≤1	S
233	≥128	R	≥128	R	32	R	8	S	8	I	4	I	≤1	S
236	16	S	16	S	4	S	2	S	2	S	0.5	S	2	S
238	8	S	16	S	4	S	2	S	2	S	≤0.25	S	≤1	S
242	8	S	8	S	4	S	2	S	1	S	≤0.25	S	≤1	S
243	16	S	8	S	4	S	2	S	1	S	≤0.25	S	≤1	S
272	64	R	64	R	8	S	4	S	2	S	1	S	≤1	S
274	16	S	8	S	4	S	2	S	2	S	0.5	S	≤1	S
278	≤4	S	≤4	S	2	S	≤1	S	2	S	≤0.25	S	≤1	S
282	≤4	S	8	S	≤1	S	≤1	S	2	S	1	S	≤1	S
285	≤4	S	≤4	S	≤1	S	≤1	S	2	S	1	S	≤1	S
289	≤4	S	8	S	≤1	S	≤1	S	2	S	1	S	≤1	S
290	8	S	8	S	≤1	S	≤1	S	2	S	1	S	≤1	S
296	≥128	R	64	R	4	S	8	S	1	S	1	S	≥16	R
298	≥128	R	≥128	R	≥64	R	8	S	8	I	4	I	≤1	S
301	16	S	8	S	4	S	2	S	2	S	0.5	S	≤1	S
302	8	S	8	S	4	S	≤1	S	2	S	0.5	S	≤1	S
309	16	S	8	S	4	S	4	S	2	S	1	S	≤1	S
310	32	R	16	S	4	S	4	S	2	S	1	S	≤1	S
312	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.

Table A4

Phenotypic resistance of P. aeruginosa strains. Data are missing for strains 198, 218 and 312 due to loss during subcultivation. MIC = minimum inhibitory concentration. N.a. = value missing due to loss of strain or failed reaction.

Sample ID	Ciprofloxacin		Moxifloxacin		Aztreonam		Amikacin		Tobramycin		Fosfomycin		Colistin
	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation
17	≤0.25	S	1	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
22	≤0.25	S	1	R	2	I	≤2	S	≤1	S	128	R	1	S
32	≤0.25	S	2	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
69	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
81	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	≥256	R	≤0.5	S
82	≤0.25	S	1	R	16	I	≤2	S	≤1	S	≤16	R	≤0.5	S
88	2	R	≥8	R	32	R	≤2	S	≤1	S	128	R	≤0.5	S
99	≤0.25	S	2	R	16	I	≤2	S	≤1	S	128	R	≤0.5	S
106	2	R	1	R	8	I	≤2	S	≤1	S	≥256	R	≤0.5	S
114	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
137	≤0.25	S	1	R	16	I	≤2	S	≤1	S	≥256	R	≤0.5	S
144	≤0.25	S	1	R	16	I	≤2	S	≤1	S	≥256	R	≤0.5	S
147	≤0.25	S	2	R	4	I	8	S	≤1	S	≥256	R	≤0.5	S
149	≤0.25	S	1	R	16	I	≤2	S	≤1	S	≥256	R	≤0.5	S
153	≤0.25	S	2	R	4	I	≤2	S	≤1	S	≤16	R	≤0.5	S
154	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	≥256	R	≤0.5	S
157	≤0.25	S	1	R	16	I	≤2	S	≤1	S	≥256	R	≤0.5	S
160	1	R	≥8	R	≥64	R	16	I	≤1	S	64	R	≤0.5	S
162	0.5	S	2	R	32	R	4	S	≤1	S	128	R	≤0.5	S
180	≤0.25	S	1	R	16	I	≤2	S	≤1	S	≥256	R	≤0.5	S
183	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	≥256	R	≤0.5	S
186	≤0.25	S	1	R	16	I	≤2	S	≤1	S	32	R	≤0.5	S
190	≤0.25	S	1	R	16	I	4	S	≤1	S	32	R	≤0.5	S
195	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	64	R	≤0.5	S
196	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	64	R	≤0.5	S
198	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
204	≤0.25	S	1	R	2	I	≤2	S	≤1	S	32	R	2	S
208	≤0.25	S	1	R	8	I	≤2	S	≤1	S	128	R	≤0.5	S
218	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.
229	≤0.25	S	1	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
233	≤0.25	S	2	R	16	I	≤2	S	≤1	S	≤16	R	≤0.5	S
236	≤0.25	S	1	R	16	I	8	S	≤1	S	64	R	≤0.5	S
238	≤0.25	S	2	R	8	I	≤2	S	≤1	S	≤16	R	≤0.5	S
242	≤0.25	S	1	R	8	I	≤2	S	≤1	S	128	R	≤0.5	S
243	≤0.25	S	2	R	16	I	≤2	S	≤1	S	64	R	≤0.5	S
272	≤0.25	S	2	R	32	R	≤2	S	≤1	S	≥256	R	≤0.5	S
274	≤0.25	S	2	R	16	I	≤2	S	≤1	S	≥256	R	≤0.5	S
278	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
282	≤0.25	S	1	R	2	I	≤2	S	≤1	S	128	R	≤0.5	S
285	≤0.25	S	1	R	2	I	≤2	S	≤1	S	128	R	≤0.5	S
289	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
290	≤0.25	S	0.5	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
296	≥4	R	≥8	R	32	R	≤2	S	≥16	R	64	R	≤0.5	S
298	≤0.25	S	2	R	16	I	≤2	S	≤1	S	≤16	R	≤0.5	S
301	≤0.25	S	2	R	16	I	≤2	S	≤1	S	≤16	R	≤0.5	S
302	≤0.25	S	1	R	4	I	≤2	S	≤1	S	128	R	≤0.5	S
309	≤0.25	S	1	R	4	I	≤2	S	≤1	S	≤16	R	≤0.5	S
310	≤0.25	S	2	R	16	I	≤2	S	≤1	S	128	R	≤0.5	S
312	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.

Table A5

Phenotypic resistance of the Klebsiella strains. MIC = minimum inhibitory concentration. ESBL = signal in phenotypic testing for extended-spectrum beta-lactamases.

Sample ID	ESBL	Ampicillin		Ampicillin/Sulbactam		Piperacillin/Tazobactam		Cefuroxime		Cefuroxime Axetil		Cefpodoxime		Cefotaxime		Ceftrazidime
		MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation
44	negative	≥32	R	16	R	≤4	S	4	I	4	I	≤0.25	S	≤1	S	≤1	S
60	negative	≥32	R	16	R	≤4	S	≤1	I	≤1	S	≤0.25	S	≤1	S	≤1	S
73	positive	≥32	R	≥32	R	≥128	R	≥64	R	≥64	R	≥8	R	≥64	R	8	R
100	positive	≥32	R	≥32	R	≥128	R	≥64	R	≥64	R	≥8	R	≥64	R	16	R
102	negative	≥32	R	≤2	I	8	S	8	I	8	I	≤0.25	S	≤1	S	≤1	S
124	negative	≥32	R	≤2	I	≤4	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S
146	negative	≥32	R	≤2	I	8	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S
177	positive	≥32	R	≥32	R	≥128	R	2	I	2	S	≤0.25	S	≤1	S	≤1	I
181	negative	≥32	R	≤2	I	≤4	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S
184	negative	16	R	≤2	I	≤4	S	2	I	2	S	≤0.25	S	≤1	S	≤1	S
199	positive	≥32	R	≥32	R	8	R	≥64	R	≥64	R	≥8	R	≥64	R	16	R
214	negative	≥32	R	≤2	I	≤4	S	≤1	I	≤1	S	≤0.25	S	≤1	S	≤1	S
217	negative	≥32	R	≥32	R	≥128	R	4	I	4	I	≤0.25	S	≤1	S	≤1	S
220	negative	≥32	R	≤2	I	≤4	S	4	I	4	I	≤0.25	S	≤1	S	≤1	S
234	negative	≥32	R	≤2	I	≤4	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S
240	negative	≥32	R	≤2	I	≤4	S	≤1	I	≤1	S	≤0.25	S	≤1	S	≤1	S
248	negative	≥32	R	≤2	I	≤4	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S
252	negative	≥32	R	16	R	≤4	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S
267	positive	≥32	R	≥32	R	32	R	≥64	R	≥64	R	≥8	R	≥64	R	16	R
277	negative	≥32	R	≥32	R	64	R	2	I	2	I	≤0.25	S	≤1	S	≤1	S
279	negative	≥32	R	≤2	I	≤4	S	2	I	2	I	≤0.25	S	≤1	S	≤1	S

Table A6

Phenotypic resistance of the Klebsiella strains. MIC = minimum inhibitory concentration. ESBL = signal in phenotypic testing for extended-spectrum beta-lactamases.

Sample ID	ESBL	Ertapenem		Imipenem		Meropenem		Gentamicin		Ciprofloxacin		Moxifloxacin		Tigecycline		Trimethoprim/Sulfamethoxazole
		MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation
44	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≤20	S
60	negative	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	1	R	2	R	≤0.5	S	≥320	R
73	positive	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	1	R	2	R	≤0.5	S	≥320	R
100	positive	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	≥4	R	≥8	R	≤0.5	S	≥320	R
102	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	0.5	R	4	R	≤20	S
124	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≤20	S
146	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	1	S	≤20	S
177	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	2	I	≥320	R
181	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≤20	S
184	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≤20	S
199	positive	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
214	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
217	negative	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	≤0.25	S	≤0.25	S	2	I	≥320	R
220	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	0.5	R	1	S	≤20	S
234	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	0.5	R	1	S	≤20	S
240	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≤20	S
248	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	2	I	≤20	S
252	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	1	S	≥320	R
267	positive	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
277	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	1	S	≥320	R
279	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≤20	S

Table A7

Phenotypic resistance of Escherichia coli strains. MIC = minimum inhibitory concentration. ESBL = signal in phenotypic testing for extended-spectrum beta-lactamases.

Sample ID	ESBL	Ampicillin		Ampicillin/Sulbactam		Piperacillin/Tazobactam		Cefuroxime		Cefuroxime Axetil		Cefpodoxime		Cefotaxime		Ceftrazidime
		MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation
41	positive	≥32	R	≥32	R	64	R	≥64	R	≥64	R	≥8	R	≥64	R	16	R
49	negative	≥32	R	16	R	≤4	S	4	I	4	S	≤0.25	S	≤1	S	≤1	S
68	negative	≥32	R	16	R	≤4	S	≤1	I	≤1	S	≤0.25	S	≤1	S	≤1	S
117	negative	≥32	R	≥32	R	64	R	4	I	4	S	≤0.25	S	≤1	S	≤1	S
152	negative	≥32	R	≥32	R	≥128	R	4	I	4	S	0.5	S	≤1	S	≤1	S
176	negative	≥32	R	≥32	R	≤4	I	2	I	2	S	≤0.25	S	≤1	S	≤1	S
221	negative	≥32	R	≥32	R	≤4	I	2	I	2	S	≤0.25	S	≤1	S	≤1	S
222	negative	≥32	R	≥32	R	≤4	I	4	I	4	S	≤0.25	S	≤1	S	≤1	S
225	positive	≥32	R	≥32	R	≤4	R	≥64	R	≥64	R	≥8	R	≥64	R	16	R
245	positive	≥32	R	≥32	R	16	I	16	R	16	R	1	S	2	I	≤1	S
270	positive	≥32	R	16	R	≤4	R	≥64	R	≥64	R	≥8	R	≥64	R	≥64	R
299	negative	≥32	R	≤2	I	≤4	S	4	I	4	S	≤0.25	S	≤1	S	≤1	S

Table A8

Phenotypic resistance of Escherichia coli strains. MIC = minimum inhibitory concentration. ESBL = signal in phenotypic testing for extended-spectrum beta-lactamases.

Sample ID	ESBL	Ertapenem		Imipenem		Meropenem		Gentamicin		Ciprofloxacin		Moxifloxacin		Tigecycline		Trimethoprim/Sulfamethoxazole
		MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation	MIC	Interpretation
41	positive	≤0.5	S	≤0.25	S	≤0.25	S	≥16	R	≥4	R	≥8	R	≤0.5	S	≥320	R
49	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
68	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≥4	R	≥8	R	≤0.5	S	≥320	R
117	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
152	negative	≤0.5	S	≤0.25	S	≤0.25	S	2	S	1	R	2	R	≤0.5	S	≥320	R
176	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
221	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
222	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≤0.25	S	≤0.25	S	≤0.5	S	≥320	R
225	positive	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	0.5	I	1	R	≤0.5	S	≥320	R
245	positive	≤0.5	S	0.5	S	≤0.25	S	≥16	R	≥4	R	≥8	R	≤0.5	S	≥320	R
270	positive	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	≥4	R	≥8	R	≤0.5	S	≤20	S
299	negative	≤0.5	S	≤0.25	S	≤0.25	S	≤1	S	0.5	I	2	R	≤0.5	S	≥320	R

In the present study, phylogenetically identical or almost identical isolates also carried the same resistomes. All E. coli strains harbored acquired beta-lactamase genes with the majority coding for small spectrum beta-lactamases such as blaTEM-1 or blaOXA-1. Only four strains carried the gene for an ESBL, in all cases blaCTX-M-15. Among the K. pneumoniae complex strains, two belonged to the species K. variicola, one to the species K. quasipneumoniae and the remaining to the species K. pneumoniae sensu stricto as reflected by intrinsic blaLEN, blaOKP and blaSHV-1 like, respectively. Genes coding for ESBL (blaCTX-M-15) were found solely in four out of 18 K. pneumoniae sensu stricto strains that also displayed resistance to oxyimino cephalosporins. In addition, several K. pneumoniae complex strains harbored blaTEM-1, single strains also contained blaOXA-1 and blaSCO-1.

With respect to P. aeruginosa, only one strain harbored acquired beta-lactamase genes (blaTEM-1 and blaSCO-1). Increased minimum inhibitory concentrations (MICs) for carbapenems as observed in some P. aeruginosa strains were neither explained by matching acquired carbapenemase genes nor by full sequence analysis of the oprD gene. The associated amino acid sequences are shown in Figure A1. As indicated, the complete oprD gene was found in all 49 P. aeruginosa isolates; there was no evidence of protein truncation by premature stop of translation. The 49 isolates could be divided into 7 subgroups according to the protein sequence of the oprD protein, which differ in a total of 30 individual amino acid exchanges and in a single 12aa/10aa-stretch. Therefore, genotypic assessment could not identify the reason for the single carbapenem-resistant P. aeuroginosa isolate 088 (ST 1682).

Figure A1

Clustal omega multiple alignment of oprD proteins—one example for the 7 detected subgroups.

Other frequently detected resistance genes in P. aeruginosa were the fosfomycin resistance gene fosA, the chloramphenicol resistance gene catB7, the aminoglycoside resistance gene aph(3′)-IIb and the fluoroquinolone-resistance gene crpP. In the Klebsiella pneumoniae complex isolates, single amino acid exchanges and the fosmomycin resistance gene fosA were frequent. Various fluoroquinolone resistance genes and disinfectant tolerance mediating genes also quantitatively dominated. Finally, a broad spectrum of acquired genes causing resistance to the assessed classes of antimicrobial drugs and tolerance to disinfectants was observed in the E. coli strains.

2.3. Identified Molecular Virulence Mechanisms

Table 2 summarizes the analysis of virulence-related genes in E. coli (without genes mediating enteropathogenicity). Data for P. aeruginosa and K. pneumoniae are presented in Table A9 and Table A10.

Table 2

Analysis of virulence determinants, ordered by strain and MLST type, of the assessed E. coli isolates. ST = Sequence type.

Sample ID	ST-Type	Pathogenicity Factor Groups
		Adherence	Invasion	Toxin	Immune Evasion	Iron Uptake	Protease
041	ST 2	fdeC	aslA, ompA			entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG,
049	ST 3		aslA, kpsC, kpsD, kpsE, kpsF, kpsM, kpsU, kpsS-like, ompA			chuS, chuU, chuV, chuW, chuY, entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG
068	ST 632		ompA			entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG
117	ST 4		aslA, kpsC, kpsD, kpsE, kpsF, kpsM, kpsU; kpsS-like, ompA	hlyB, hlyC, hlyD,	tcpC	chuA, chuS, chuT, chuU, chuV, chuW, chuX, chuY, entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG, hlyA, iroN,	pic, sat, vat
152	ST 22	sfaB, sfaC, sfaD, sfaE, sfaF, sfaG, sfaH, sfaS, sfaX, sfaY	aslA, kpsC, kpsD, kpsE, kpsF, kpsM, kpsU; kpsS-like, ompA	cnf1; hlyA, hlyB, hlyC, hlyD,	tcpC	chuA, chuS, chuT, chuU, chuV, chuW, chuX, chuY, entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG, iroN,	vat
176	ST 132		aslA, kpsC, kpsD, kpsE, kpsF, kpsM, kpsU; kpsS-like, ompA			entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG,	sat
221	ST 132		aslA, kpsC, kpsD, kpsE, kpsM, kpsU; kpsS-like, ompA			entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG,	sat
222	ST 132		aslA, kpsC, kpsD, kpsE, kpsF, kpsM, kpsU; kpsS-like, ompA			entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG,	sat
225	ST 506		aslA, kpsC, kpsD, kpsE, kpsF, kpsM, kpsU; kpsS-like, ompA			chuA, chuS, chuT, chuU, chuV, chuW, chuX, chuY, entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepC, fepD, fepG,	sat
245	ST 2		aslA, ompA			entA-like, entB, entC, entE, entF, entS, fepB, fepC, fepD, fepG
270	ST 2		aslA, ompA			entA-like, entB, entC, entE, entS, fepA, fepB, fepC, fepD, fepG
299	ST 1018		ompA			entA-like, entB, entC, entE, entF, entS, fepA, fepB, fepD, fepG

Table A9

Analysis of virulence determinants, ordered by strain and MLST type, of the assessed P. aeruginosa isolates. ST = Sequence type.

Sample ID	ST-Type	Pathogenicity Factor Groups
		Adherence	Anti-Phagocytosis	Biosurfactant	Iron Uptake	Pigment	Protease	Toxin	Regulation	Secretion System
017	ST 381	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algP/algR3 algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdD, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
022	ST 2483	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
032	ST 3587	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
069	ST 360	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
081	ST 244	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW like, xcpX, xcpY, xcpZ
082	ST 514	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
088	ST 1682	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
099	ST 244	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
106	ST 1521	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdD, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
114	ST 244	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW like, xcpX, xcpY, xcpZ
137	ST 3014	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
144	ST 245	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
147	ST 245	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
149	ST 381	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimV, pilA, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
153	ST 704	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	plcH	lasI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
154	ST 244	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW like, xcpX, xcpY, xcpZ
157	ST 2616	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilA, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
160	ST 170	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilA, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
162	ST 274	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilA, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
180	ST 856	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
183	ST 244	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW like, xcpX, xcpY, xcpZ
186	ST 3588	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
190	ST 871	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
195	ST 988	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
196	ST 2475	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
198	ST 2476	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
204	ST 639	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
208	ST 132	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
218	ST 856	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
229	ST 270	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdD, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
233	ST 3227	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
236	ST 266	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
238	ST 3589	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	plcH	lasI, rhlI	xcpP, xcpQ, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
242	ST 3590	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3 like, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
243	ST 3590	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
272	ST 2033	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
274	ST 2033	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
278	ST 988	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
282	ST 554	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
285	ST 554	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilA like, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
289	ST 1485	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdD, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
290	ST 1485	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdD, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
296	ST 235	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW like, xcpX, xcpY, xcpZ
298	ST 3227	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
301	ST 3593	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
302	ST 1755	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
309	ST 3592	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimT, fimU, fimV, pilB, pilD, pilE, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, pilX pilY1, pilY2, pilC, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdE	phzM, phzS	aprA, lasA	plcH	lasI, rhlI	xcpP, xcpQ, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
310	ST 532	waaA, waaC, waaF, waaG, waaP, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, fpvA pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA	phzM, phzS	aprA lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ
312	ST 381	waaA, waaC, waaF, waaG, waaP, wzy, wzz, chpA, chpB, chpC, chpD, chpE, fimV, pilB, pilD, pilF, pilG, pilH, pilI, pilK, pilM, pilN, pilO, pilP, pilQ, pilR, pilS, pilT, pilU, pilC like, xcpA/pilD	alg44, alg8, algA, algB, algC, algD, algE, algF, algG, algI, algJ, algK, algL, algP/algR3, algQ, algR, algU, algW, algX, algZ, mucA, mucB, mucC	rhlA, rhlB	fptA, pchA, pchB, pchC, pchD, pchE, pchF, pchG, pchH, pchI, pchR, pvdA, pvdD, pvdE	phzM, phzS	aprA, lasA	toxA, plcH	lasI, rhlI	xcpP, xcpQ, xcpR, xcpS, xcpT, xcpU, xcpV, xcpW, xcpX, xcpY, xcpZ

Table A10

Analysis of virulence determinants, ordered by strain and MLST type, of the assessed K. pneumoniae isolates. ST = Sequence type.

Sample ID	ST-Type	Pathogenicity Factor Groups
		Adherence	Biofilm Formation	Efflux Pump	Immune Evasion	Iron Uptake	Nutritional Factor	Regulation	Secretion System	Serum Resistance	Toxin
044	ST 327	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza like, wzi	entA, entB, entC, entD, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE like		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
060	ST 5379	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB	impA/tssA, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC
073	ST 39	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA, sciN/tssJ, tle1, tli1, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
100	ST 152	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza like, wzi	entA, entB, entC, entD, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG
102	ST 514	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
124	ST 399	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkF, mrkH, mrkJ	acrA, acrB	cpsACP, galF, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB		glf, wbbM, wbbN, wbbO, wzm, wzt
146	ST 4	fimA, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
177	ST 17	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza like, wzi	entA, entB, entC, entD, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
181	ST 5380	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gmd like, gnd, manB, manC, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB,	impA/tssA, sciN/tssJ, tle1, tli1, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
184	ST 5381	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza like, wzi	entA, entB, entC, entD like, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE like		rcsA, rcsB	impA/tssA, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC
199	ST 17	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB like, manC, ugd, wza, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC
214	ST 6	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkB, mrkC, mrkD, mrkF, mrkH, mrkI,	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
217	ST 3154	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
220	ST 5382	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB like, manC, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE,	allA, allB, allC, allD, allR, allS	rcsA, rcsB
234	ST 109	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB,	impA/tssA, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
240	ST 5383	fimC, fimD, fimF, fimG, fimH, fimI, fimK	mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB		glf, wbbM, wbbN, wbbO, wzm, wzt
248	ST 5384		mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB	vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
252	ST 607	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, wza like, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB	sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
267	ST 36	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		rcsA, rcsB	impA/tssA, sciN/tssJ, tle1, tli1, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
277	ST 530	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, manB, manC, ugd, wza, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB	impA/tssA like, sciN/tssJ, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	glf, wbbM, wbbN, wbbO, wzm, wzt
279	ST 5385	fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, fimK	mrkA, mrkB, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ	acrA, acrB	cpsACP, galF, gnd, ugd, wza, wzi	entA, entB, entC, entE, entF, fepA, fepB, fepC, fepD, fepG, fes, ybdA, iroE		rcsA, rcsB,	impA/tssA, sciN/tssJ, tle1, tli1, tssF, tssG, vasE/tssK, vgrG/tssI, vipA/tssB, vipB/tssC	wzm, wzt

The virulence-associated gene exoU, which has been described in association with the P. aeruginosa high-risk clone ST 135 [25], was recorded three times, associated with ST 135 (sample ID 296), ST 532 (sample ID 310) and ST 2483 (sample ID 22), respectively. Based on a Kleborate assessment, a positive virulence score was calculated for 7 out of 21 K. pneumoniae strains, comprising the known high-risk clones ST 17 (sample IDs 177, 199) and ST 152 (sample ID 100) [26], next to the clones ST 4 (sample ID 146), ST 6 (sample ID 214), ST 36 (sample ID 267) and ST 39 (sample ID 73), respectively. With focus on some important virulence associated genes in Klebsiella spp., ybt genes were detected in the abovementioned 7 samples, iroE was recorded in all 21 strains, while clb or rpmA genes were not detected.

Iron-uptake-related genes were numerous in all analyzed bacterial strains. For P. aeruginosa and K. pneumoniae, various secretion system-associated genes were found. Immune evasion-related genes were highly abundant in K. pneumoniae but not in E. coli isolates. Adherence-related genes were numerous in P. aeruginosa and in K. pneumoniae but not in E. coli.

Numerous invasion-associated genes were detected in E. coli, antiphagocytosis-associated genes were found in P. aeruginosa, and biofilm-associated genes in K. pneumoniae.

Less frequently detected were: toxin genes in E. coli and K. pneumoniae, protease genes in E. coli and P. aeruginosa, regulation genes in P. aeruginosa and K. pneumoniae, biosurfactant and pigment genes in P. aeruginosa and nutrition factor, efflux pumps and serum resistance genes in K. pneumoniae.

3. Discussion

Within this study, we aimed at filling information gaps on the molecular epidemiology of Gram-negative bacteria from chronic infected wounds in rural Ghana. Phylogenetic analyses based on core genome comparison indicated a high clonal diversity of the wound-associated isolates. Clonal clusters were restricted to nine P. aeruginosa clusters and one E. coli cluster, most likely indicating nosocomial transmission, which has most likely occurred in the wound dressing room that patients’ visit on a weekly basis.

ST 135 and ST 244, which are among the worldwide top 10 P. aeruginosa high-risk clones [25], were found among the P. aeruginosa wound isolates. In detail, one ST 135 Pseudomonas aeruginosa isolate was detected, carrying the beta-lactamase-encoding genes blaTEM-1B and blaSCO-1 and an exoU gene, next to five ST 244 without acquired beta-lactamases. Focusing on known pathogenic K. pneumoniae clones [26], two ST 17 strains, a clone reported to be associated with carbapenem-resistance, and one ST 152 strain, a clone known from the Caribbean as common carrier of multiple resistance genes, were detected. Strains carrying the ybt and iro genes were also identified as high-risk clones by the Kleborate software. From the observed E. coli ST types, none have been previously reported as being associated with pathogenic clones so far [27].

In line with the phenotypical antibiotic resistance results previously published [23], numerous acquired resistance determinants were detected in the bacterial strains under investigation. Focusing on the few observed clusters, comparable resistome compositions point towards recent nosocomial transmission. The gene blaCTX-M-15 was identified as the determinant of the detected extended spectrum beta-lactamase (ESBL) expression in ESBL positive Enterobacterales [23]. This is in line with previous reports from both human and livestock-associated ESBL positive Enterobacterales in Ghana [28,29,30,31,32,33,34]. In P. aeruginosa and K. pneumoniae, blaSCO-1, which has initially been described from an Acinetobacter baumannii isolate from Argentina [35], was observed. Beta-lactamases with high hydrolytic effects on carbapenems were lacking, the same applies to protein truncation by premature stop of translation of the oprD gene in P. aeruginosa. Accordingly, the genetic background of carbapenem resistance of a single P. aeruginosa strain could not be resolved, although downregulation of oprD expression due to mutations outside of the gene or ampC (class C betalactamase) overexpression could not be excluded as likely reasons.

Substance-specific genes and genes encoding efflux pumps mediating tolerance to disinfectants were observed in Enterobacterales. Therefore, further monitoring of the spread of disinfectant tolerance-associated genes and the effects of their abundance on disinfectant-based skin and wound decolonization strategies [36] seem advisable.

The importance of highly abundant virulence factors like iron-uptake- and secretion system-related genes in P. aeruginosa is comprehensively described in the literature [37,38]. Other genes reported in the literature like regulation-associated virulence genes, recently reported, were less frequently observed in our isolates [39,40]. However, due to lacking information on the individual etiological relevance of each isolate, any association with clinical effects remains speculative.

Further limitations of this study include a rather small sample size and the lack of a comparison strain collection containing isolates from other clinical specimens and environmental strains. Accordingly, the interpretation of the etiological relevance of individual strains remains challenging and is clearly beyond the scope of this work.

In summary, a broad spectrum of Gram-negative clones was isolated from the chronic wounds of the Ghanaian patients. Thereby, known high-risk clones [25,26,27] played only a minor role. Observed resistance patterns and mechanisms were in line with the spectrum expected from previous reports [23,28,29,30,31,32,33,34].

4. Materials and Methods

4.1. Sample Collection, Bacterial Culture and Antibiotic Susceptibility Testing

Single patient strains of P. aeruginosa, E. coli and K. pneumoniae complex were isolated from patients ≥15 years with an infected chronic wound at the Outpatient Department (OPD) of the Agogo Presbyterian Hospital, in the Asante Akim North District of rural Ghana. Patients typically visit the wound dressing room of the OPD on a weekly basis. Sampling was performed from January 2016 to November 2016. Sample collection and microbiological investigations were reported previously [23]. Antibiotic susceptibility was tested by the disk diffusion method and interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines v.6.0 (http://www.eucast.org (accessed on 15 January 2016)). Bacterial strains and antibiotic susceptibility were confirmed using the VITEK2 System. Those data have been published before [23].

4.2. DNA Isolation and Whole Genome Sequencing

Bacterial DNA was isolated using the MasterPure Complete DNA and RNA Purification Kit (LGC standards GmbH, Wesel, Germany) and sent for whole genome sequencing (WGS) to BGI Europe, Denmark, Copenhagen. A BGISEQ-500 device was used for sequencing, generating 2 × 150 bp paired-end reads with an aimed coverage of 100×. Original raw data were upload for public use to the short-read archive (SRA, NCBI) under the accession number PRJNA699140. Details on the strain-specific SRA accession numbers are provided in Table A11.

Table A11

Details on the strain-specific short-read archive (SRA) accession numbers.

Sample ID	Percentage of Good Targets (SeqSphere+)	Average Coverage (Assembled) (SeqSphere+)	Approximated Genome Size (Megabases) (SeqSphere+)	Species (Kraken2)	Sequence Type	Complex Type (SeqSphere+)	SRA Accession
Iso00017	99.4	105	6.7	Pseudomonas aeruginosa	381	1791	SRR13617317
Iso00022	99.4	102	6.9	Pseudomonas aeruginosa	2483	1792	SRR13617316
Iso00032	99.2	106	6.6	Pseudomonas aeruginosa	3587	1793	SRR13617305
Iso00041	99.4	97	5.0	Escherichia coli	2 (Pasteur)	11349	SRR13617294
Iso00044	99.7	116	5.1	Klebsiella pneumoniae	327	5462	SRR13617283
Iso00049	98.7	94	5.2	Escherichia coli	3 (Pasteur)	11350	SRR13617272
Iso00060	99.6	112	5.3	Klebsiella pneumoniae	5379	5463	SRR13617261
Iso00068	99.6	109	4.9	Escherichia coli	632 (Pasteur)	11351	SRR13617250
Iso00069	99.6	104	6.8	Pseudomonas aeruginosa	360	1794	SRR13617239
Iso00073	99.4	104	5.8	Klebsiella pneumoniae	39	5464	SRR13617236
Iso00081	98.7	108	6.6	Pseudomonas aeruginosa	244	1795	SRR13617315
Iso00082	99.4	112	6.3	Pseudomonas aeruginosa	514	1796	SRR13617314
Iso00088	97.8	105	6.8	Pseudomonas aeruginosa	1682	1797	SRR13617313
Iso00099	99.4	106	6.6	Pseudomonas aeruginosa	244	1798	SRR13617312
Iso00100	99.2	108	5.5	Klebsiella pneumoniae	152	5465	SRR13617311
Iso00102	99.2	111	5.4	Klebsiella pneumoniae	514	5466	SRR13617310
Iso00106	99.5	110	6.4	Pseudomonas aeruginosa	1521	1799	SRR13617309
Iso00114	99.4	105	6.7	Pseudomonas aeruginosa	244	1800	SRR13617308
Iso00117	99.2	95	5.3	Escherichia coli	4 (Pasteur)	11352	SRR13617307
Iso00124	99.4	112	5.3	Klebsiella pneumoniae	399	5467	SRR13617306
Iso00137	99.4	110	6.4	Pseudomonas aeruginosa	3014	1801	SRR13617304
Iso00144	99.6	109	6.5	Pseudomonas aeruginosa	245	1802	SRR13617303
Iso00146	99.4	110	5.5	Klebsiella pneumoniae	4	5468	SRR13617302
Iso00147	99.5	108	6.6	Pseudomonas aeruginosa	245	1802	SRR13617301
Iso00149	99.6	104	6.9	Pseudomonas aeruginosa	381	1803	SRR13617300
Iso00152	99.4	98	5.2	Escherichia coli	22 (Pasteur)	11353	SRR13617299
Iso00153	98.5	111	6.4	Pseudomonas aeruginosa	704	?	SRR13617298
Iso00154	99.4	102	7.0	Pseudomonas aeruginosa	244	1805	SRR13617297
Iso00157	99.6	114	6.3	Pseudomonas aeruginosa	2616	1806	SRR13617296
Iso00160	99.2	115	6.2	Pseudomonas aeruginosa	170	1807	SRR13617295
Iso00162	99.1	111	6.5	Pseudomonas aeruginosa	274	1808	SRR13617293
Iso00176	99.0	98	5.1	Escherichia coli	132 (Pasteur)	11354	SRR13617292
Iso00177	99.6	108	5.5	Klebsiella pneumoniae	17	5469	SRR13617291
Iso00180	99.8	110	6.5	Pseudomonas aeruginosa	856	1809	SRR13617290
Iso00181	99.9	107	5.6	Klebsiella pneumoniae	5380	5470	SRR13617289
Iso00183	99.5	107	6.7	Pseudomonas aeruginosa	244	1795	SRR13617288
Iso00184	98.3	104	5.6	Klebsiella variicola subsp. variicola	5381	5471	SRR13617287
Iso00186	98.7	113	6.3	Pseudomonas aeruginosa	3588	1810	SRR13617286
Iso00190	99.7	114	6.3	Pseudomonas aeruginosa	871	1811	SRR13617285
Iso00195	99.5	111	6.5	Pseudomonas aeruginosa	988	1812	SRR13617284
Iso00196	99.5	101	7.1	Pseudomonas aeruginosa	2475	1813	SRR13617282
Iso00198	99.6	112	6.4	Pseudomonas aeruginosa	2476	1814	SRR13617281
Iso00199	99.4	108	5.6	Klebsiella pneumoniae	17	5472	SRR13617280
Iso00204	99.5	104	6.9	Pseudomonas aeruginosa	639	1815	SRR13617279
Iso00208	99.7	109	6.5	Pseudomonas aeruginosa	132	1816	SRR13617278
Iso00214	99.7	108	5.5	Klebsiella pneumoniae	6	5473	SRR13617277
Iso00217	99.8	104	5.7	Klebsiella pneumoniae	3154	5474	SRR13617276
Iso00218	99.7	109	6.5	Pseudomonas aeruginosa	856	1809	SRR13617275
Iso00220	97.8	110	5.4	Klebsiella quasipneumoniae subsp. similipneumoniae	5382	5475	SRR13617274
Iso00221	99.0	94	5.1	Escherichia coli	132 (Pasteur)	11354	SRR13617273
Iso00222	99.0	96	5.1	Escherichia coli	132 (Pasteur)	11354	SRR13617271
Iso00225	99.1	99	5.2	Escherichia coli	506 (Pasteur)	11355	SRR13617270
Iso00229	99.6	109	6.5	Pseudomonas aeruginosa	270	1817	SRR13617269
Iso00233	97.8	114	6.1	Pseudomonas aeruginosa	3227	1818	SRR13617268
Iso00234	99.7	111	5.5	Klebsiella pneumoniae	109	5476	SRR13617267
Iso00236	99.7	112	6.4	Pseudomonas aeruginosa	266	1819	SRR13617266
Iso00238	98.7	108	6.6	Pseudomonas aeruginosa	3589	1820	SRR13617265
Iso00240	98.9	112	5.4	Klebsiella pneumoniae	5383	5477	SRR13617264
Iso00242	98.9	111	6.4	Pseudomonas aeruginosa	3590	1821	SRR13617263
Iso00243	98.9	111	6.4	Pseudomonas aeruginosa	3590	1821	SRR13617262
Iso00245	99.3	107	4.8	Escherichia coli	2 (Pasteur)	11356	SRR13617260
Iso00248	97.2	108	5.5	Klebsiella quasivariicola	5384	5478	SRR13617259
Iso00252	99.6	112	5.3	Klebsiella pneumoniae	607	5479	SRR13617258
Iso00267	99.6	103	5.7	Klebsiella pneumoniae	36	5480	SRR13617257
Iso00270	99.2	100	4.9	Escherichia coli	2 (Pasteur)	11358	SRR13617256
Iso00272	99.5	109	6.5	Pseudomonas aeruginosa	2033	1822	SRR13617255
Iso00274	99.4	109	6.5	Pseudomonas aeruginosa	2033	1822	SRR13617254
Iso00277	99.4	109	5.5	Klebsiella pneumoniae	530	5481	SRR13617253
Iso00278	99.6	110	6.5	Pseudomonas aeruginosa	988	1823	SRR13617252
Iso00279	99.7	111	5.5	Klebsiella pneumoniae	5385	5482	SRR13617251
Iso00282	99.3	108	6.6	Pseudomonas aeruginosa	554	1824	SRR13617249
Iso00285	99.3	109	6.5	Pseudomonas aeruginosa	554	1824	SRR13617248
Iso00289	99.6	112	6.3	Pseudomonas aeruginosa	1485	1825	SRR13617247
Iso00290	99.7	113	6.3	Pseudomonas aeruginosa	1485	1825	SRR13617246
Iso00296	99.7	106	6.7	Pseudomonas aeruginosa	235	1826	SRR13617245
Iso00298	97.8	116	6.1	Pseudomonas aeruginosa	3227	1818	SRR13617244
Iso00299	99.3	108	4.6	Escherichia coli	1018 (Pasteur)	11357	SRR13617243
Iso00301	98.6	112	6.3	Pseudomonas aeruginosa	3593	1827	SRR13617242
Iso00302	99.6	113	6.3	Pseudomonas aeruginosa	1755	1828	SRR13617241
Iso00309	98.6	109	6.5	Pseudomonas aeruginosa	3592	1829	SRR13617240
Iso00310	99.3	105	6.8	Pseudomonas aeruginosa	532	1830	SRR13617238
Iso00312	99.4	106	6.7	Pseudomonas aeruginosa	381	1791	SRR13617237

4.3. Whole Genome Sequencing and Data Analysis

All raw data passed quality control using FASTQC v.0.11.4 [41] and were used for further analysis. Taxonomic classification and contamination check of raw-reads was performed using KRAKEN2 v.2.0.8-beta [42]. Phylogenetic analysis based on core genome multi locus sequence typing (cgMLST) analysis was performed using the commercial software SeqSphere+ v. 7.2.0 (Ridom GmbH, Münster, Germany) [43]. The software pipeline included assessment of read data and adapter control using FASTQC followed by genome assembly using the internally provided assembler Velvet, applying default settings. The reference genomes NC_000913.3 (E. coli), NC_002516.2 (P. aeruginosa) and NC_01273.1 (K. pneumoniae species complex) were used for cgMLST analyses. Only samples with a ration of “good cgMLST targets” higher than 90% were included in the phylogenetic analysis. Novel cgMLST-based complex types (CT) were automatically assigned by the SeqSphere software. Unknown alleles and profiles of MLST genes were submitted to pubmlst.org or Institute Pasteur to establish novel sequence types (ST). Isolates were defined to be clonally identical with allele differences less than four. Moreover, raw data were assembled with SPAdes v3.13.11 [44] using the careful option. Scaffolds shorter than 500 bp or with a coverage smaller than ten were sorted out, using an in-house script. Abricate v.0.9.9 [45] was used to screen for resistance and virulence genes in SPAdes assembly files, using NCBI AMRFinderPlus [46] and VFDB [47] as reference databases (both updated 6 November 2020), respectively. Additionally, SPAdes assemblies were uploaded to ResFinder4.1 [48] to obtain WGS predicted phenotypes against different antimicrobials by using default settings (%ID > 90, minimum length > 60%) and to Kleborate to predict virulence genes in Klebsiella isolates.

4.4. Ethical Considerations

The Committee on Human Research, Publications and Ethics, School of Medical Science, Kwame Nkrumah University of Science and Technology in Kumasi, Ghana, approved this study (approval number CHRPE/AP/078/16).

5. Conclusions

In conclusion, this study provides a molecular insight into the epidemiology of Gram-negative bacteria isolated from chronic wound infections from patients in rural Ghana. Epidemiological data that focus on the distribution and spread of antimicrobial resistance determinants and associated virulence factors in resource-limited settings are scarce. Although the study is a small cross-sectional assessment, which cannot replace continuous surveillance programs, it might provide a glimpse of prevailing Gram-negative bacteria isolated from wound infections in this area of Ghana. Considering the ongoing need for resistance and virulence surveillance in tropical regions, larger future studies are desirable.