Emergence of multidrug resistance and extensive drug resistance among enterococcal clinical isolates in Egypt

Figures & data

Table 1 List of primers used in this study

Target gene	Primer pair sequence	Product size (bp)	Annealing temp (°C)	Reference
rrs (16S rRNA)	GGATTAGATACCCTGGTAGTCC TCGTTGCGGGACTTAACCCAAC	320	54	^Citation24
ddlE. faecalis	ATCAAGTA CAGTTAGTCT ACGATTCAAAGCTAACTG	941	54	^Citation24
ddlE. faecium	TTGAGGCAGACCAGATTGACG TATGACAGCGACTCCGATTCC	658	54	^Citation24
vanA	CATGAATAGAATAAAAGTTGCAATA CCCCTTTAACGCTAATACGATCAA	1,030	54	^Citation24
vanB	AAGCTATGCAAGAAGCCATG CCGACAATCAAATCATCCTC	536	54	^Citation23
vanC1	GGTATCAAGGAAACCTC CTTCCGCCATCATAGCT	822	54	^Citation24
vanC2/C3	CGGGGAAGATGGCAGTAT CGCAGGGACGGTGATTTT	484	54	^Citation24
aac(6’)-Ie-aph(2’’)-Ia	CCAAGAGCAATAAGGGCATA CACTATCATAACCACTACCG	200	52	^Citation31
ant(6’)-Ia	ACTGGCTTAATCAATTTGGG GCCTTTCCGCCACCTCACCG	597	52	^Citation30
blaZ	ACTTCAACACCTGCTGCTTTC TGACCACTTTTATCAGCAACC	173	50	^Citation31
tet(K)	TTATGGTGGTTGTAGCTAGAAA AAAGGGTTAGAAACTCTTGAAA	370	52	^Citation34
tet(L)	GTMGTTGCGCGCTATATTCC TGAAMGRWAGCCCACCTAA	715	52	^Citation34
tet(M)	ACAGAAAGCTTATTATATAAC TGGCGTGTCTATGATGTTCAC	170	52	^Citation32
ermA	TCTAAAAAGCATGTAAAAGAA CTTCGATAGTTTATTAATATTAGT	644	52	^Citation35
ermB	GAAAAGGTACTCAACCAAATA AGTAACGGTACTTAAATTGTTTAC	639	52	^Citation35
ermC	TCAAAACATAATATAGATAAA GCTAATATTGTTTAAATCGTCAAT	642	52	^Citation35
msrA/B	GCAAATGGTGTAGGTAAGACAACT ATCATGTGATGTAAACAAAAT	400	52	^Citation35
cfr	TGTATGTTTTGACTTTCGGCACCGG ATTATCTTCCACCCAGTAGTCC	1,320	52	^Citation36
optrA	AGGTGGTCAGCGAACTAA ATCAACTGTTCCCATTCA	1,395	55	^Citation37
int-Tn	GCGTGATTGTATCTCACT GACGCTCCTGTTGCTTCT	1,046	50	^Citation33
xis-Tn	AAGCAGACTGAGATTCCTA GCGTCCAATGTATCTATAA	194	50	^Citation33

Table 2 Antibiotic susceptibility testing of enterococcal clinical isolates

Antimicrobial agent	E. faecalis, 71 (68.9%)			E. faecium, 32 (31.1%)			Total, 103 (100%)
	R (%)	I (%)	S (%)	R (%)	I (%)	S (%)	R (%)	I (%)	S (%)
Ampicillin	21 (29.6)	0 (0)	50 (70.4)	32 (100)	0 (0)	0 (0)	53 (51.5)	0 (0)	50 (48.5)
Ampicillin/sulbactam	21 (29.6)	0 (0)	50 (70.4)	31 (96.9)	1 (3.1)	0 (0)	52 (50.5)	1 (1)	50 (48.5)
Amoxicillin	19 (26.8)	2 (2.8)	50 (70.4)	30 (93.8)	2 (6.3)	0 (0)	49 (47.6)	4 (3.9)	50 (48.5)
Amoxicillin/clavulanic acid	19 (26.8)	2 (2.8)	50 (70.4)	30 (93.8)	2 (6.3)	0 (0)	49 (47.6)	4 (3.9)	50 (48.5)
Imipenem	15 (21.1)	0 (0)	56 (78.9)	30 (93.8)	2 (6.3)	0 (0)	45 (43.7)	2 (1.9)	56 (54.4)
Meropenem	19 (26.8)	3 (4.2)	49 (69)	31 (96.9)	1 (3.1)	0 (0)	50 (48.5)	4 (3.9)	49 (47.6)
HLGR	57 (80.3)	0 (0)	14 (19.7)	25 (78.1)	0 (0)	7 (21.9)	82 (79.6)	0 (0)	21 (20.4)
HLSR	17 (24)	0 (0)	54 (76.1)	21 (65.6)	0 (0)	11 (34.4)	38 (36.9)	0 (0)	65 (63.1)
Erythromycin	66 (93)	5 (7)	0 (0)	28 (87.5)	4 (12.5)	0 (0)	94 (91.3)	9 (8.7)	0 (0)
Clarithromycina	40 (88.9)	0 (0)	5 (11.1)	21 (80.8)	0 (0)	5 (19.2)	61 (85.9)	0 (0)	10 (14)
Azithromycina	35 (77.8)	1 (2.2)	9 (20)	18 (69.2)	0 (0)	8 (30.8)	53 (74.6)	1 (1.4)	17 (24)
Clindamycin	71 (100)	0 (0)	0 (0)	32 (100)	0 (0)	0 (0)	103 (100)	0 (0)	0 (0)
Ciprofloxacin	60 (84.5)	4 (5.6)	7 (9.9)	24 (75)	0 (0)	8 (25)	84 (81.6)	4 (3.9)	15 (14.5)
Levofloxacin	63 (88.7)	1 (1.4)	7 (9.9)	29 (90.6)	1 (3.1)	2 (6.3)	92 (89.3)	2 (2)	9 (8.7)
Tertracycline	66 (93)	4 (5.6)	1 (1.4)	30 (93.8)	2 (6.3)	0 (0)	96 (93.2)	6 (5.8)	1 (1)
Doxycycline	33 (46.5)	25 (35.2)	13 (18.3)	24 (75)	7 (21.9)	1 (3.1)	57 (55.3)	32 (31.1)	14 (13.6)
Vancomycin	1 (1.4)	4 (5.6)	66 (93)	6 (18.8)	5 (15.6)	21 (65.6)	7 (6.8)	9 (8.7)	87 (84.5)
Linezolid	4 (5.6)	0 (0)	67 (94.4)	6 (18.8)	0 (0)	26 (81.3)	10 (9.7)	0 (0)	93 (90.3)

Note:

a Susceptibility for clarithromycin and azithromycin was performed on 71 isolates only.

Abbreviations: HLGR, high-level gentamicin resistance; HLSR, high-level streptomycin resistance.

Figure 1 Genotyping of E. faecalis clinical isolates using RAPD-PCR method.

Notes: Dendrogram was created using RAPD-PCR patterns of E. faecalis clinical isolates. Similarity clustering analysis was carried out using UPGMA and Dice coefficient. The dashed line is hypothetical, indicating 85% similarity. Antibiotic susceptibility, resistance determinants, and biofilm formation capacity among E. faecalis clinical isolates were reported. Antibiotic susceptibility: Black cell: Resistant, Gray cell: Intermediate, White cell: Sensitive. Biofilm formation: White cell: Biofilm Non-producer, Light gray cell: Weak Biofilm Producer, Dark gray cell: Moderate Biofilm Producer, Black cell: Strong Biofilm Producer. Resistance determinants: Black cell: Resistance gene detected, White cell: Resistance gene not detected.

Abbreviations: AM, Ampicillin; AMC, Amoxicillin/clavulanic acid; AX, Amoxicillin; AZM, Azithromycin; CIP, ciprofloxacin; CLR, Clarithromycin; DA, Clindamycin; DO, Doxycline; E, Erythromycin; HLGR, High Level Gentamicin Resistance; HLSR, High Level Streptomycin Resistance; IPM, Imipenem; LEV, Levofloxacin; LZD, Linezolid; MEM, Meropenem; ND, Not Determined; RAPD-PCR, random amplified polymorphic DNA-PCR; SAM, Ampicillin/Sulbactam; TE, Tetracycline; UPGMA, unweighted-pair group method with arithmetic averages; VA, Vancomycin.

Figure 2 Genotyping of E. faecium clinical isolates using RAPD-PCR method.

Notes: Dendrogram was created using RAPD-PCR patterns of E. faecium clinical isolates. Similarity clustering analysis was carried out using UPGMA and Dice coefficient. The dashed line is hypothetical, indicating 85% similarity. Antibiotic susceptibility, resistance determinants, and biofilm formation capacity among E. faecalis clinical isolates were reported. Antibiotic susceptibility: Black cell: Resistant, Gray cell: Intermediate, White cell: Sensitive. Biofilm formation: White cell: Biofilm Non-producer, Light gray cell: Weak Biofilm Producer, Dark gray cell: Moderate Biofilm Producer, Black cell: Strong Biofilm Producer. Resistance determinants: Black cell: Resistance gene detected, White cell: Resistance gene not detected.

Abbreviations: AM, Ampicillin; AMC, Amoxicillin/clavulanic acid; AX, Amoxicillin; AZM, Azithromycin; CIP, Ciprofloxacin; CLR, Clarithromycin; DA, Clindamycin; DO, Doxycline; E, Erythromycin; HLGR, High Level Gentamicin Resistance; HLSR, High Level Streptomycin Resistance; IPM, Imipenem; LEV, Levofloxacin; LZD, Linezolid; MEM, Meropenem; ND, Not Determined; RAPD-PCR, random amplified polymorphic DNA-PCR; SAM, Ampicillin/Sulbactam; TE, Tetracycline; UPGMA, unweighted-pair group method with arithmetic averages; VA, Vancomycin.

Figure S1 Agarose gel electrophoresis for detection of antimicrobials resistance determinants among enterococcal isolates. (A). Detection of antimicrobials resistance encoding genes among enterococcal isolates by PCR. Lane M is Molecular weight marker. 1:aac(6’)-Ie-aph(2’’)-Ia (200 bp); 2: ant(6’)-Ia (597 bp); 3: tetK (370 bp); 4: tetL (715 bp); 5: tetM (170 bp); 6: ermB (639 bp) and 7: msrA/B (400 bp); 8: optrA (1395 bp); 9: int-Tn (1046 bp); and 10: xis-Tn (194 bp) genes that are used for detection antimicrobials resistance among enterococcal isolates. (B). Multiplex PCR for detection of vancomycin resistance determinants (vanA(1030 bp),vanB(536 bp),vanC1 (822 bp), and vanC_2/3 (484 bp)), E. faecium-specific (658 bp), E. faecalis-specific (941 bp) and of rrs (320 bp) genes. Lane M is Molecular weight marker. 1:E. faecalis isolate; 2:E. faecium isolate; 3:E. faecium vanB isolate; 4: E. faecalis vanC1 isolate; 5: E. faecium vanC1isolate; 6:E. faecalis vanBisolate.

Table S1 Prevalence of antibiotic susceptibility among enterococcal species isolated from different clinical sources

Antimicrobial agent	E. faecalis(N=71)			E. faecium(N=32)			Pb-value
	Ra (%)	I (%)	S (%)	R (%)	I (%)	S (%)
Ampicillin	21 (29.6)	0 (0)	50 (70.4)	32 (100)	0 (0)	0 (0)	The chi-square statistic is 41.0091. The P-value is <0.00001.
Ampicillin/sulbactam	21 (29.6)	0 (0)	50 (70.4)	31 (96.9)	1 (3.1)	0 (0)	The chi-square statistic is 40.0396. The P-value is <0.00001.
Amoxicillin	19 (26.8)	2 (2.8)	50 (70.4)	30 (93.8)	2 (6.3)	0 (0)	The chi-square statistic is 41.158. The P-value is <0.00001.
Amoxicillin/clavulanic acid	19 (26.8)	2 (2.8)	50 (70.4)	30 (93.8)	2 (6.3)	0 (0)	The chi-square statistic is 41.158. The P-value is <0.00001.
Imipenem	15 (21.1)	0 (0)	56 (78.9)	30 (93.8)	2 (6.3)	0 (0)	—
Meropenem	19 (26.8)	3 (4.2)	49 (69)	31 (96.9)	1 (3.1)	0 (0)	—
HLGR	57 (80.3)	0 (0)	14 (19.7)	25 (78.1)	0 (0)	7 (21.9)	The chi-square statistic is 0.3891. The P-value is 0.823203.
HLSR	17 (24)	0 (0)	54 (76.1)	21 (65.6)	0 (0)	11 (34.4)	The chi-square statistic is 16.6831. The P-value is 0.000238.
Erythromycin	66 (93)	5 (7)	0 (0)	28 (87.5)	4 (12.5)	0 (0)	The chi-square statistic is 1.1451. The P-value is 0.564093.
Clarithromycinc	40 (88.9)	0 (0)	5 (11.1)	21 (80.8)	0 (0)	5 (19.2)	The chi-square statistic is 1.0435. The P-value is 0.593476.
Azithromycinc	35 (77.8)	1 (2.2)	9 (20)	18 (69.2)	0 (0)	8 (30.8)	The chi-square statistic is 1.0791. The P-value is 0.583011.
Clindamycin	71 (100)	0 (0)	0 (0)	32 (100)	0 (0)	0 (0)	The chi-square statistic is 0.6366. The P-value is 0.727381.
Ciprofloxacin	60 (84.5)	4 (5.6)	7 (9.9)	24 (75)	0 (0)	8 (25)	The chi-square statistic is 3.9361. The P-value is 0.139728.
Levofloxacin	63 (88.7)	1 (1.4)	7 (9.9)	29 (90.6)	1 (3.1)	2 (6.3)	The chi-square statistic is 0.6724. The P-value is 0.714478.
Tertracyclin^e	66 (93)	4 (5.6)	1 (1.4)	30 (93.8)	2 (6.3)	0 (0)	The chi-square statistic is 0.3255. The P-value is 0.8498.
Doxycyclin^e	33 (46.5)	25 (35.2)	13 (18.3)	24 (75)	7 (21.9)	1 (3.1)	The chi-square statistic is 8.2472. The P-value is 0.016186.
Vancomycin	1 (1.4)	4 (5.6)	66 (93)	6 (18.8)	5 (15.6)	21 (65.6)	The chi-square statistic is 14.2318. The P-value is 0.000812.
Linezolid	4 (5.6)	0 (0)	67 (94.4)	6 (18.8)	0 (0)	26 (81.3)	The chi-square statistic is 4.628. The P-value is 0.098863.

Notes:

a R: Resistant; I: Intermediate resistance; S: Sensitive.

b E.faecalis vs E. faecium;P-value<0.05 was considered statistically significant.

c Susceptibility for clarithromycin and azithromycin was performed on 71 isolates only.

Abbreviations: HLGR, High Level Gentamicin Resistance; HLSR, High Level Streptomycin Resistance.

Table S2 Prevalence of biofilm formation among enterococcal isolates collected from different clinical sources

	No. (%) of enterococcal isolates with biofilm formation capacity					Pa-value
	Strong	Moderate	Weak	Biofilm producers	Biofilm non-producers
E. faecalis (N=71)	13 (18.3)	29 (40.9)	24 (33.8)	66 (93)	5 (7)	The chi-square statistic
E. faecium (N=32)	8 (25)	12 (37.5)	11 (34.4)	31 (96.9)	1 (3.1)	is 1.1294. The P-value is
Total (N=103)	21 (20.4)	41 (39.8)	35 (34)	97 (94.2)	6 (5.8)	0.769975.

Note:

a E. faecalis vs E. faecium; P-value<0.05 was considered statistically significant.

Table S3 Correlation between biofilm formation capacity and antibiotic susceptibility among enterococcal clinical isolates

	AM		SAM		AX		AMC		IPM		MEM		VA		HLGR		HLSR
	R (53)	S (50)	R (53)	S (50)	R (53)	S (50)	R (53)	S (50)	R (47)	S (56)	R (54)	S (49)	R (16)	S (87)	R (82)	S (21)	R (38)	S (65)
S_BP	15	6	15	6	15	6	15	6	12	9	12	9	7	14	17	4	11	10
M_BP	17	24	17	24	17	24	17	24	16	25	19	22	7	34	30	11	13	28
W_BP	20	15	20	15	20	15	20	15	16	19	20	15	2	33	29	6	11	24
N_BP	1	5	1	5	1	5	1	5	3	3	3	3	0	6	6	0	3	3
chi^Citation2 P	8.353 0.039254		8.353 0.039254		8.353 0.039254		8.353 0.039254		1.889 0.595689		1.122 0.771695		8.593 0.034407		1.383 0.709473		3.529 0.317045
	TE		DO		CIP		LEV		E		DA		AZM		CLR		LZD
	R (102)		S (1)		R (89)		S (14)		R (88)		S (15)		R (94)		S (9)		R (103)
S_BP	21		0		19		2		16		5		18		3		21
M_BP	40		1		37		4		36		5		38		3		41
W_BP	35		0		29		6		30		5		33		2		35
N_BP	6		0		4		2		6		0		5		1		6
chi^Citation2 P	2.466 0.481511		3.176 0.365237		1.504 0.681259		1.79 0.617422		2.511 0.473275		2.511 0.473275		5.254 0.154128		0.848 0.837894		8.760 0.032654

Note: P-value<0.05 was considered statistically significant.

Abbreviations: AM, Ampicillin; AMC, Amoxicillin/clavulanic acid; AX, Amoxicillin; AZM, Azithromycin; CIP, Ciprofloxacin; CLR, Clarithromycin; DA, Clindamycin; DO, Doxycycline; E, Erythromycin; HLGR, High Level Gentamicin resistance; HLSR, High Level Streptomycin resistance; IPM, Imipenem; LEV, Levofloxacin; LZD, Linezolid; M_BP, Moderate biofilm producer; MEM, Meropenem; N_BP, Non-biofilm producer; R, Resistant; S, Sensitive; S_BP, Strong biofilm producer; SAM, Ampicillin/sulbactam; TE, Tetracycline; VA, Vancomycin; W_BP, Weak biofilm producer.

Table S4 Distribution of multidrug resistance (MDR) and extensive drug resistance (XDR) among enterococcal clinical isolates

E. faecalis (n=71)		E. faecium (n=32)
Resistance profile	No. (%) of isolates	Resistance profile	No. (%) of isolates
G, S, A, C, F, T, V, L	2 (2.8)	G, S, A, F, T, V, L	4 (12.5)
G, S, A, C, F, T, L	1 (1.4)	G, A, F, T, V, L	1 (3.1)
G, S, A, C, F, T	4 (5.6)	G, S, A, F, T, V	4 (12.5)
G, A, C, F, T	9 (12.7)	G, S, A, F, T	11 (34.4)
G, A, F, T, V	1 (1.4)	G, A, T, V, L	1 (3.1)
G, S, C, F, T	1 (1.4)	G, A, F, T	3 (9.4)
G, S, F, T, V	1 (1.4)	A, F, T, V	1 (3.1)
G, S, F, T	5 (7)	G, A, S, F	1 (3.1)
G, C, F, T	3 (4.2)	S, A, F, T	1 (3.1)
G, A, F, T	1 (1.4)	A, F, T	5 (15.6)
G, S, A, F	1 (1.4)
A, C, F, T	1 (1.4)
G, F, T	18 (25.4)
G, S, F	2 (2.8)
C, F, T	1 (1.4)
A, F, T	1 (1.4)
F, T, L	1 (1.4)
F, T	8 (11.3)
G, F	6 (8.5)
F, V	1 (1.4)
G	2 (2.8)
—	1 (1.4)

Abbreviations: A, Penicillins (Ampicillin); C, Carbapenems (Imipenem or Meropenem); F, Fluoroquinolones (Ciprofloxacin or Levofloxacin); G, High Level Gentamicin resistance; L, Oxazolidinones (Linezolid); S, High Level Streptomycin resistance; T, Tetracyclines (Doxycycline); V, Glycopeptides (Vancomycin).

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