Emergence of a dalbavancin induced glycopeptide/lipoglycopeptide non-susceptible Staphylococcus aureus during treatment of a cardiac device-related endocarditis

Figures & data

Antimicrobial susceptibility profiles of the four isolated S. aureus strains. All MICs were obtained by use of E-tests, except for dalbavancin where a broth microdilution assay was performed additionally

	DR-I1	DR-I2	DR-I3	DR-I4
Oxacillin	0.500	2.000	2.000	3.000
Cefoxitin	4.000	6.000	6.000	8.000
Vancomycin	1.500	1.500	2.000	2.000
Teicoplanin	0.190	0.380	2.000	16.000
Dalbavancin	0.047	0.047	0.190	0.380
Dalbavancin (BMA)^a	0.063	0.063	0.500	1.000
Daptomycin	0.380	0.380	0.500	0.750
Trimethoprim/Sulfamethoxazole (E-test)	0.064	0.047	0.064	0.064
Fusidic acid	>256.000	0.064	0.250	0.190
Doxycyclin	0.250	0.047	0.190	0.190
Gentamicin	0.500	0.190	0.032	0.064
Clindamycin	0.125	0.047	0.047	0.032
Linezolid	2.000	1.000	2.000	2.000
Ciprofloxacin	0.750	0.500	0.190	0.250
Fosfomycin	1.500	4.000	0.500	0.500
Rifampicin	0.016	0.006	0.094	0.064

BMA Broth microdilution assay

^aDalbavancin broth microdilution assay was performed according to a recently published study and to CLSI and EUCAST convenients^44,45. S. aureus ATCC 29213 was used as reference strain and yielded a MIC of 0.063 mg/L

Oligonucleotides used for PCR analysis

Gene	Forward oligonucleotide (5′–3′)	Reverse oligonucleotide (5′–3′)
Spa^a	TAA AGA CGA TCC TTC GGT GAGC	CAGCAGTAGTGCCGTTTGCTT
Arc^b	TTG ATT CAC CAG CGC GTA TTG TC	AGG TAT CTG CTT CAA TCA GCG
Aro^b	ATC GGA AAT CCT ATT TCA CAT TC	GGT GTT GTA TTA ATA ACG ATA TC
Glpf^b	CTA GGA ACT GCA ATC TTA ATC C	TGG TAA AAT CGC ATG TCC AAT TC
Gmk^b	ATC GTT TTA TCG GGA CCA TC	TCA TTA ACT ACA ACG TAA TCG TA
Pta^b	GTT AAA ATC GTA TTA CCT GAA GG	GAC CCT TTT GTT GAA AAG CTT AA
Tpi^b	TCG TTC ATT CTG AAC GTC GTG AA	TTT GCA CCT TCT AAC AAT TGT AC
yqil^b	CAG CAT ACA GGA CAC CTA TTG GC	CGT TGA GGA ATC GAT ACT GGA AC

Spa Staphylococcal protein A, arc carbamate kinase, aroe shikimate dehydrogenase, glpf glycerol kinase, gmk guanylate kinase, pta phosphate acetyltransferase, tpi triosephosphate isomerase, yqil acetyle coenzyme A acetyltransferase

^aOligonucleotides for spa typing

^bOligonucleotides for MLST

Genetic differences among the four consecutively obtained isolates

Mutation (nucleotide no. in DR-I1)	Locus^a	Gene name	Gene function	Effect of mutation	Presentation
					DR-I1	DR-I2	DR-I3	DR-I4
G to A (136230)	jcf180000001136	pbp2	penicillin binding protein	G146R	−	−	+	+
T to G (26436)	Jcf7180000001176	rpoB	DNA-directed RNA polymerase subunit beta	R436H	−	−	+	+
Deletion A (112755)	jcf7180000001168	stp1	serine/threonine protein kinase Stk1	frameshift	−	+	+	+
Insertion A (30413)	jcf7180000001177		hypothetical protein SA_21	frameshift	−	−	+	+
Deletion (40976–40440)	jcf7180000001177		DHH domain of GdpP^b	534 bp frameshift	−	−	+	+
Deletion (207587–218096)	jcf7180000001135		permease WP_061734446.1	10.509 bp	−	−	+	+
			CAAX amino protease WP_000801979.1		−	−	+	+
			pathogenicity island protein AKJ49778.1		−	−	+	+
			phage capsid protein WP_085065008.1		−	−	+	+
			DNA primase WP_031905599.1		−	−	+	+
			XRE family transcriptional regulator WP_000583241.1		−	−	+	+
			integrase BAM28662.1		−	−	+	+
C to T (33629)	jcf7180000001176	fusA	translation elongation factor G	H475Y	+	−	−	−
CAAAAGAAAGCTAAG to TGTAAATGGCGGTTA (90560)	jcf7180000001168	16S rRNA	modified 3′-end of 30S ribosomal protein S16	Altered 3′ end	−	−	+	+

^aGene identification number in the first susceptible isolate DR-I1

^bGGDEF domain protein containing phosphodiesterase

Fig. 1 Time history of antimicrobial usage and bacterial isolation

Fig. 2 Ultrastructural analysis of dalbavancin non-susceptibility.

Scanning electron micrographs at a magnification of ×5000 (a, b, c) and ×10,000 (d, e, f), as well as transmission electron micrographs at a magnification of ×30,000 (g, h, i). White arrows indicate dividing bacterial cells

Fig. 3 Relative numbers of bacterial cells in specific cell cycle stages.

a Relative numbers of bacterial cells per morphologic cell cycle stage presented as % of 200 counted cells. b Exemplary pictures of irregular cell division observed in the two isolates DR-I3 and DR-I4

Koeth, L. M., DiFranco-Fisher, J. M. & McCurdy, S. A Reference broth microdilution method for dalbavancin in vitro susceptibility testing of bacteria that grow aerobically. J. Vis. Exp. 103, e53028 (2015).

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Patel, J. B. et al. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically 10th Ed. (CLSI, Wayne, PA, 2015).

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