Phenotype-Genotype Characterization and Antibiotic-Resistance Correlations Among Colonizing and Infectious Methicillin-Resistant Staphylococcus aureus Recovered from Intensive Care Units

Figures & data

Table 1 List of Primers Used in the Current Study and Predicted Size of PCR Products

Gene	Forward Primer (5ʹ to 3ʹ)	Reverse Primer (5ʹ to 3ʹ)	Size	Reference
arcC	TTG ATT CAC CAG CGC GTA TTG TC	AGG TAT CTG CTT CAA TCA GCG	456bp	(Enright, et al, 2000)
aroE	ATC GGA AAT CCT ATT TCA CAT TC	GGT GTT GTA TTA ATA ACG ATA TC	456bp	(Enright, et al, 2000)
cfr	GACTTTCGGCACCGGTAAT	CAGTAGTCCATTCATATTTG	745bp	(Shore, et al, 2010)
glpF	CTA GGA ACT GCA ATC TTA ATC C	TGG TAA AAT CGC ATG TCC AAT TC	465bp	(Enright, et al, 2000)
gmk	ATC GTT TTA TCG GGA CCA TC	TCA TTA ACT ACA ACG TAA TCG TA	430bp	(Enright, et al, 2000)
Luk-pvl	ATCATTAGGTAAAATGTCTGGACATGATCCA	GCATCAAGTGTATTGGATAGCAAAAGC	430bp	(Zhang, et al, 2004)
mecA	GTAGAAATGACTGAACGTCCGATAA	CCAATTCCACATTGTTTCGGTCTAA	310bp	(Zhang, et al, 2004)
pta	GTT AAA ATC GTA TTA CCT GAA GG	GAC CCT TTT GTT GAA AAG CTT AA	475bp	(Enright, et al, 2000)
tpi	TCGTTCATTCTGAACGTCGTGAA	TTTGCACCTTCTAACAATTGTAC	402bp	(Enright, et al, 2000)
yqiL	CAGCATACAGGACACCTATTGGC	CGTTGAGGAATCGATACTGGAAC	516bp	(Enright, et al, 2000)
ERIC 1	ATGTAAGCTCCTGGGGATTCAC			(Candan, et al, 2013)

Figure 1 Distribution of isolates according to symptoms manifestation. Pie charts showing (A) the relative distribution of colonizing and infectious isolates, as well as (B) the detailed sites of isolation of both types.

Figure 2 Distribution of biofilm formation ability among both colonizing and infectious isolates. (A) A stacked bar plot indicating the percentage of colonizing isolates with biofilm and non biofilm forming ability. (B) A stacked bar plot indicating the percentage of infectious isolates with biofilm and non biofilm forming ability. The X-axis represents the percentage of either biofilm forming isolates (dark grey bars) or non biofilm forming isolates (light grey bar). The number written inside each bar represents the number of isolates constituting the final percentage.

Figure 3 Minimum inhibitory concentration patterns. (A) Comparison of linezolid MICs of both colonizing and infectious isolates. (B) Comparison of vancomycin MICs of both colonizing and infectious isolates. Grey bars represent colonizing isolates and black bars representing infectious ones.

Figure 4 Antibiogram of the tested colonizing and infectious isolates. (A) Colonizing isolates antibiogram results tabular representation towards the 16 tested antibiotics. (B) Infectious isolates antibiogram results tabular representation towards the 16 tested antibiotics; classification was done as follows “resistant (R), intermediate (I) and sensitive (S)”. (C) Bar charts showing the percentages of resistant isolates towards the 16 different antibiotics used. The black bars represent the infectious isolates, and the grey bars represent the colonizing isolates.

Abbreviations: VAN, vancomycin; LZD, linezolid; AMK, amikacin; MEM, meropenem; SXT, trimethoprim/sulfamethoxazole; ENX, erythromycin; RIF, rifampicin; AMX, amoxicillin clavulanic; DOX, doxycycline; CLX, clindamycin; FD, fusidic; LVX, levofloxacin; MUP, mupirocin; FOX, cefoxitin; TEC, teicoplanin; QD, quinupristin-dalfopristin.

Figure 5 ERIC-PCR analysis of the tested MRSA isolates. Unweighted pair group method using arithmetic overage algorithm (UPGMA) clustering method, showing the genetic similarity among MRSA isolates by enterobacterial repetitive intergenic consensus (ERIC) genotyping. Radial dendrogram, in decreasing nodes order, was generated with FigTree v1.4.4. Blue labels represent the colonizing isolates and black labels represent the infectious isolates.

Table 2 MLST Results Using MLST Database (Bold), BLAST, or Both (Bold and Underlined)

Isolates	MLST Genes
	arcC	aroE	glpF	gmk	pta	tpi	yqiL
Colonized-1(11)	CA-347 99%	No match	CA-347 98%	Allele 6	Allele 14 100%	FORC_001, 99%	FORC_001, 99% Allele 2 2 diff
Colonized-2 (15)	Allele 13 100%	Allele 13 100%	Allele 93 4 diff	Strain 79, 99% Allele 1 1 diff	Strain 502A 100% Allele 12 3 diff	Strain 79, 97% Allele 209	Allele 13 99%
Colonized-3 (19)	Staphylococous aureus subsp. aureus strain FORC_001, complete genome
Infectious-1 (26)	FORC_001 99% Allele 2 100%	Strain 27b, MRSA 99% Allele 3 100%	Strain 79, s10 genome 98% Allele 93 3 diff	No match	Strain 27b, MRSA 99% Allele 4 100%	No match	Strain 27b, MRSA 99% Allele 3 100%
Infectious-2 (34)	S. aureus strain FORC_001 100% Allele 2 100%	Strain 27b MRSA 99% Allele 3 99%	Strain 79, 97% Allele 93 3 diff	Strain 27b MRSA 100% Allele 1 100%	Strain 27b MRSA 100% Allele 4 100%	Strain 27b MRSA 97% Allele 154 100%	Strain 27b MRSA 99% Allele 3 100%
Infectious-3 (37)	S. aureus strain FORC_001 100% Allele 2 100%	Strain 27b MRSA 99%	Strain KIBGE-MB99 99% Allele 93 8 diff	Strain 79, 100% Allele 1 100%	Strain 27b MRSA 100% Allele 4 100%	No match	Strain 27b MRSA 99% Allele 3 100%

Figure 6 Phenotype-genotype correlation. Phenotype-genotype correlation matrix. A colored matrix representing the correlations between all possible pairs of phenotypes (isolate type, source, specimen, biofilm ability, and antibiogram) and tested genes of the whole isolates collection. Vancomycin and linezolid were not included as all isolates were sensitive to them. The color represents Pearson’s correlation coefficient and its intensity represents the coefficient’s value (shades of blue are positive correlations and shades of orange-brown are negative correlations). Figure labels for antibiotics; figure labels for biofilm. Score; strong biofilm forming=3, moderate biofilm forming=2, weak biofilm forming=3. Figure labels for minimum inhibitory concentration (MIC); LZD_Status= borderline or not borderline MIC and LZD_MIC=1, 2 and 4 µg/mL. The figure was generated using R-software.

Abbreviations: LZD, linezolid; AMK, amikacin; MEM, meropenem; SXT, trimethoprim sulfamethoxazole; ENX, erythromycin; RIF, rifampicin; AMX, amoxicillin clavulanic; DOX, doxycycline; CLX, clindamycin; FD, fusidic; LVX, levofloxacin; MUP, mupirocin; FOX, cefoxitin; TEC, teicoplanin; QD, quinupristin-dalfopristin.