Fluoroquinolone-Resistant Sequence Type 131 Subgroups O25b and O16 Among Extraintestinal Escherichia coli Isolates from Community-Acquired Urinary Tract Infections

Abstract

The multidrug-resistant sequence type 131 (ST131) Escherichia coli is a spreading epidemiological burden particularly among isolates resistant to fluoroquinolones. We aimed to evaluate the commonality of ST131-O25b and ST131-O16 among fluoroquinolone-resistant E. coli isolates causing community-acquired urinary tract infections (UTIs) at Fayoum University Hospital, in Egypt. Ninety-two fluoroquinolone-resistant E. coli isolates were subjected to multiplex PCR for detection of ST131 of either O25b or O16 subgroups. Positive isolates were then assessed for antimicrobial susceptibility and virulence genotyping. Out of 92 fluoroquinolone-resistant E. coli isolates, 56 (60.9%) isolates were O25b/O16 subgroups of ST131, including 44 (78.6%) ST131-O25b and 12 (21.4%) ST131-O16 subgroups. All the O25b/O16 ST131 isolates were sensitive to meropenem, where ST131-O25b isolates were significantly more resistant to extended spectrum cephalosporins compared to S131-O16 strains. All the O25b/O16 ST131 isolates harbored three or more of the virulence factors associated with extraintestinal pathogenic E. coli status. ST131-O16 showed a significantly higher virulence score than ST131-O25b isolates. Our results bring to highlight the emergence of O25b/O16 ST131 isolates between community acquired UTIs among Egyptian patients. This is the first report for the presence of O16 isolates in Egypt, showing a lower predominance than the O25b subgroup. The high prevalence of O25b/O16 ST131 isolates requires strict stewardship on antimicrobial use, notably fluoroquinolones, to control the endemicity of such emerging multidrug-resistant clone in the community.

Introduction

Urinary tract infections (UTIs) are one of the commonest bacterial infections affecting humans, with extraintestinal pathogenic Escherichia coli (ExPEC) being responsible for more than 90% of cases of community acquired UTIs.¹ Infection by ExPEC has significant morbidity, mortality, and increased healthcare expenses.² Treatment is usually complicated by the high prevalence and the extent of antimicrobial drug resistance, particularly, fluoroquinolone and trimethoprim–sulfamethoxazole resistance.³

E. coli sequence type 131 (ST131) was first identified by multilocus sequence typing as a pandemic in different continents in 2008.^4,5 ST131 strains have been recognized as a disseminated virulent E. coli lineage in both hospital and community healthcare settings from all parts of the world.^6,7

Strains of ST131 E. coli have two common antibiotic resistance traits with the majority showing resistance against fluoroquinolone, while a minority shows resistance to extended spectrum cephalosporins due to extended-spectrum β-lactamase (ESBL) production.^2,8 ESBL-negative fluoroquinolone-resistant ST131 strains represent a prominent cause of E. coli infections in many parts of the globe.^9,10

ST131 is a molecularly qualified ExPEC, harboring various virulence genes (adhesins, toxins, and siderophors), and it belongs to phylogenetic group B2 in contrast to the classical antimicrobial-resistant E. coli.^2,4,11

Although most of the ST131 isolates usually exhibit the serotype O25:H4, few reports identified a newer serotype, O16:H5,^12–14 constituting a different phylogenetic clade.¹⁵ Moreover, three more different O serogroups were identified among ST131 isolates: O19a, O136, and O153,¹⁶ in addition to the presence of some O-nontypable ST131 strains.¹⁷

Considering the very scarce reports about ST131 infections in Egypt, the present study aimed to assess the clonal commonality of the two main subgroups of ST131 E. coli UTIs in Egypt. In addition, the antimicrobial resistance pattern and the virulence genotype of both ST131-O25b and O16 subgroups among fluoroquinolone-resistant E. coli were also investigated.

Materials and Methods

Collection of samples

A total of 150 non-duplicate E. coli isolates were obtained from symptomatic community acquired UTI patients attending the outpatient Clinic at Fayoum University Hospital in the period between October 2014 and January 2015. All isolates were identified by routine microbiological methods. Isolates were anonymously collected and stored with no accompanying demographic data.

Antibiotic susceptibility testing

All isolates were examined for ciprofloxacin susceptibility by disk diffusion method, with fluoroquinolone resistance defined as non-susceptibility to ciprofloxacin. Disk diffusion susceptibility testing on Muller-Hinton agar (Oxoid) for ciprofloxacin nonsusceptibility isolates was done following the Clinical and Laboratory Standards Institute (CLSI) recommendations for antimicrobial susceptibility testing.¹⁷ The used panel of antibiotics was as follows: levofloxacin (5 μg), gatifloxacin (5 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), aztreonam (30 μg), amoxicillin/clavulanic acid (30 μg), meropenem (10 μg), amikacin (10 μg), and co-trimoxazole (25 μg) (Oxoid). Intermediate susceptibility was interpreted as resistance. The number of antibiotics (including ciprofloxacin) to which an isolate was resistant was considered as the resistance score. For each group, median and range of resistance scores were calculated. O25b/O16 ST131's proportional contribution to resistance to each antibiotic was calculated. Multidrug-resistant isolates were those resistant to more than three groups of the tested antibiotics.

Molecular methods

DNA extraction

DNA extraction was performed according to the manufacturer's instructions using GeneJET Genomic DNA Purification Kit (K0721; Thermo-Scientific).

Screening of O25b/O16 ST131

Prevalence of O25b/O16 subgroups of ST131 among fluoroquinolone-resistant E. coli isolates was molecularly detected. Clades were detected according to Johnson et al.¹² Briefly, a multiplex PCR was carried out in 50 μl reactions using 25 μl hot-start MyTaq HS Mix PCR master mix (Bio-25045; Bioline); 10 pmol of each of the following primers: pabB ST131-O25b.f, pabB ST131-O25b.r, trpAST131-O16.f, and trpAST131-O16.r (Table 1); and 2 μl of DNA. The PCR amplification was performed with initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation for 5 sec at 94°C, annealing for 20 sec at 63°C, and a final extension for 5 min at 72°C.

Table 1.

Primer Sequences, Annealing Temperature, and Expected Amplified Products

Target gene	Primer	Sequence	Annealing temperature (°C)	Reference	Amplicon size (bp)
pabB	pabBST131-O25b.f	5′-TCCAGCAGGTGCTGGATCGT-3′	63	¹¹	347
	pabBST131-O25b.r	5′-GCGAAATTTTTCGCCGTACTGT-3′
trpA	trpAST131-O16.f	5′-AAAACCGCGCCGCGTTACCT-3′	63	¹²	145
	trpAST131-O16.r	5′-CCAGAAATCGCGCCCGCATT-3′
fimH	fimH.f	5′-AACAGCGATGATTTCCAGTTTGTGTG-3	65	²⁶	465
	fimH.r	5′-ATTGCGTACCAGCATTAGCAATGTCC-3
papC	pap1	5′-GACGGCTGTACTGCAGGGTGTGGCG-3′	65	²⁷	328
	pap2	5′-ATATCCTTTCTGCAGGGATGCAATA-3′
sfa/focDE	sfa1	5′-CTCCGGAGAACTGGGTGCATCTTAC-3′	65	²⁷	410
	sfa2	5′-CGGAGGAGTAATTACAAACCTGGCA-3′
afaC	afa.f	5′-CGGCTTTTCTGCTGAACTGGCAGGC-3′	65	²⁷	672
	afa.r	5′-CCGTCAGCCCCCACGGCAGACC-3′
iutA	iutA.f	5′-GGCTGGACATCATGGGAACTGG-3′	63	²⁸	302
	iutA.r	5′-CGTCGGGAACGGGTAGAATCG-3′
sat	sat1	5′-GCAGCAAATATTGATATATCA-3′	57	²⁹	630
	sat2	5′-GTTGTTGACCTCAGCAAGGAA-3′
KpsMII	KpsMII.f	5′-GCGCATTTGCTGATACTGTTG-3′	52	²⁸	272
	KpsMII.r	5′-CATCCAGACGATAAGCATGAGCA-3′
usp	usp.f	5′-CGGCTCTTACATCGGTGCGTTG-3′	60	³⁰	615
	usp.r	5′-GACATATCCAGCCAGCGAGTTC-3′

fimH, mannose-specific adhesin of type I fimbriae; papC, P fimbriae; sfa/focDE, central region of sfaS and focG operons; afaC, afa adhesins; iutA, aerobactin siderophore receptor; sat, the secreted autotransporter toxin; kpsMII, group 2 capsule; usp, uropathogenic-specific protein.

Detection of virulence genes

The presence of eight ExPEC associated virulence genes was investigated. Adhesion encoding genes were mannose-specific adhesin of type 1 fimbriae (fimH), P fimbriae (papC), central region of sfaS and focG operons (sfa/focDE), and afa adhesins (afaC), in addition to aerobactin siderophore receptor (iutA), the secreted autotransporter toxin (sat), group 2 capsule (kpsMII), and uropathogenic-specific protein (usp). The sum of positive virulence factors for each isolate was used as the virulence score. Median and range of virulence scores were calculated for each group.

Primers used for the amplification of both ST131-O16 and ST131-O25b and the virulence genes are listed in Table 1. The primers were synthesized by Invitrogen. The PCR amplification started with denaturation at 94°C, 35 cycles of denaturation at 94°C for 2 min, annealing at a specific temperature for 30 sec (Table 1), and extension at 72°C for 1 min. The PCR products were loaded on 2% agarose gel and then were photographed under UV light.

Isolates with three or more of the tested virulence genes were identified as ExPEC.¹⁸

Data analysis

Collected data were computerized and analyzed using Statistical Package for Social Science (SPSS) version 16. Number and percentage were used to describe variables. Relationship between variables was done using chi-square test. Fisher's exact test was used for two by two tables when expected cell count was less than five. Scores were presented using median, and range comparison of scores between groups was done using Mann–Whitney U test. p-Values of less than 5% were considered statistically significant.

Ethical approval

This study has been revised and approved by the Fayoum Faculty of Medicine Research Ethics Committee.

Results

Molecular detection of E. coli O25b/O16 subgroups of ST131 clonal group

Of the 150 E. coli isolates tested for antimicrobial resistance, 92 (61.3%) were found resistant to fluoroquinolones. PCR analysis showed that the overall prevalence of O25b/O16 ST131 was 56 (60.9%) of the 92 fluoroquinolone-resistant isolates, comprising 44 (78.6%) ST131-O25b, while 12 (21.4%) were identified as ST131-O16.

Antibiotic susceptibility testing

All of the fluoroquinolone-resistant O25b/O16 ST131 isolates were sensitive to meropenem. Forty isolates (71.4%) were resistant to cefoxitin, 36 (64.3%) to ceftazidime, 40 (71.4%) to cefotaxime, 31 (55.4%) to cefepime, 34 (60.7%) to aztreonam, 39 (69.6%) to amoxicillin/clavulanic acid, 24 (42.9%) to amikacin, and 47 (83.9%) to co-trimoxazole (Table 2). Distribution of antibiotic resistance among ST131-O25b and ST131-O16 subgroups is shown in Table 2. Resistance score was lower for ST131-O16 (median score 7) (Table 2). Multidrug resistance was detected in 48 (85.7%) of O25b/O16 ST131 isolates. Proportional contributions of O25b/O16 ST131 to resistance, by agent, were 56% for cefoxitin, 53% for ceftazidime, 63% for cefotaxime, 57% for cefepime, 64% for aztreonam, 59% for amoxicillin/clavulanic acid, 43% for amikacin, and 67% for co-trimoxazole (Table 2).

Table 2.

Prevalence of Antimicrobial Resistance Among Fluoroquinolone-Resistant ST131 and Non-ST131 Escherichia coli Isolates from Community Acquired Urinary Tract Infection

	O25b/O16 ST131 isolates					p^b
	O25b strains, N = 44	O16, N = 12	Total, N = 56	Non-O25b/O16, N = 36
Antibiotic resistance ^a	n (%)	n (%)	n (%)	n (%)	Estimated contribution of O25b/O16 ST131, %	O25b vs. O16	Non-O25b/O16 vs. O25b/O16 ST131
Cefoxitin	36 (81.8)	4 (33.3)	40 (71.4)	32 (88.9)	0.56	0.002	0.069
Ceftazidime	35 (79.5)	1 (8.3)	36 (64.3)	31 (86.1)	0.53	0.000	0.014
Cefotaxime	34 (77.3)	6 (50)	40 (71.4)	25 (69.4)	0.63	0.002	0.649
Cefepime	28 (63.6)	3 (25)	31 (55.4)	24 (66.7)	0.57	0.09	0.390
Aztreonam	27 (61.4)	7 (58.3)	34 (60.7)	20 (55.6)	0.64	1.0	0.512
Amoxicillin/clavulanic acid	31 (70.5)	8 (66.6)	39 (69.6)	28 (77.8)	0.59	0.726	0.628
Amikacin	19 (43.2)	5 (41.7)	24 (42.9)	32 (88.9)	0.43	0.525	0.000
Co-trimoxazole	35 (79.5)	12 (100)	47 (83.9)	23 (63.9)	0.67	0.180	0.044
Multidrug resistant	37 (84.1)	11 (91.7)	48 (85.7)	31 (86.1)		0.180	0.759
Resistance score	10 (3–11)^c	7 (5–8)		10 (4–11)		0.001	0.154

All isolates were resistant to fluoroquinolones and meropenem.

p-Values ≤0.05 were considered statistically significant.

The value denotes mean and range.

ST, sequence type.

O25b/O16 ST131 isolates, compared with non-O25b/O16 isolates, exhibited significantly lower prevalence of resistance to ceftazidime (64.3% vs. 86.1%) (p = 0.014) and amikacin (42.9% vs. 88.9%) (p < 0.05). The only statistically significant higher resistance prevalence difference between O25b/O16 ST131 and non-O25b/O16 was for co-trimoxazole (83.9% vs. 63.9%) (p = 0.044). ST131-O25b had significantly higher prevalence of resistance compared with ST131-O16 to cefoxitin (81.8% vs. 33.3%) (p = 0.002), ceftazidime (79.5% vs. 8.3%) (p = 0.000), and cefotaxime (77.3% vs. 50%) (p = 0.002). Accordingly, resistance scores were significantly lower among ST131-O16 isolates (median 7, range 5–8) than ST131-O25b isolates (median 10, range 3–11; p = 0.001).

Virulence traits

Four different virulence traits were present in ≥90% of the ST131 isolates that included (overall prevalence; definition) the following: fimH (100%, mannose-specific adhesion of type I fimbriae), kpsMII (92.9%, group 2 capsule), usp (91.1%, uropathogenic-specific protein), and iutA (91.1%; aerobactin system) (Table 3).

Table 3.

Prevalence of Virulence Traits Among Fluoroquinolone-Resistant O25b/O16 ST131 and Non-O25b/O16 E. coli Isolates from Community Acquired Urinary Tract Infection

	O25b/O16 ST131				p^a
	O25b strains, N = 44	O16, N = 12	Total, N = 56	Non-O25b/O16, N = 36
Virulence traits ^a	n (%)	n (%)	n (%)	n (%)	O25b vs. O16	Non-O25b/O16 vs. O25b/O16 ST131
fimH	44 (100)	12 (100)	56 (100)	33 (91.7)	1	0.057
papC	5 (11.4)	5 (41.6)	10 (17.9)	16 (44.4)	0.055	0.003
sfa	9 (20.5)	5 (41.6)	13 (23.2)	13 (36.1)	0.263	0.231
afa	16 (36.4)	4 (33.3)	20 (35.7)	11 (30.6)	1.0	1.0
kpsMII	40 (90.9)	12 (100)	52 (92.9)	12 (33.3)	0.567	0.000
usp	39 (88.6)	12 (100)	51 (91.1)	7 (19.4)	0.567	0.000
sat	39 (88.6)	11 (91.7)	50 (89.3)	16 (44.4)	1.0	0.000
iutA	40 (90.9)	11 (91.7)	51 (91.1)	21 (58.3)	0.567	0.000
Virulence score	5 (3–6)^b	6 (4–7)	6 (3–7)	3 (2–6)	0.032	0.000

p-Values ≤0.05 were considered statistically significant.

The value denotes mean and range.

fimH, mannose-specific adhesin of type I fimbriae; papC, P fimbriae; sfa/focDE, central region of sfaS and focG operons; afaC, afa adhesins; iutA, aerobactin siderophore receptor; sat, the secreted autotransporter toxin; kpsMII, group 2 capsule; usp, uropathogenic-specific protein.

The O25b/O16 ST131 isolates exhibited significantly greater prevalence of the kpsMII (p < 0.001), usp (p < 0.001), sat (p < 0.001), and iutA (p < 0.001) compared with non-O25b/O16 isolates (Table 3). Conversely, they exhibited significantly lower prevalence of the papC (p = 0.003). Accordingly, the O25b/O16 ST131 isolates as a group had numerically higher virulence scores (median score 6, range 3–7) than the non-O25b/O16 isolates (median score 3, range 2–6; p < 0.001). In addition, O25b/O16 ST131 isolates highly fulfilled the molecular criteria for ExPEC than the non-O25b/O16 (100% vs. 86.2%; Table 4).

Table 4.

Prevalence of Extraintestinal Pathogenic E. coli Among Fluoroquinolone-Resistant O25b/O16 ST131 and Non-O25b/O16 E. coli Isolates from Community Acquired Urinary Tract Infection

	O25b/O16 ST131				p^a
	O25b strains, N = 44	O16, N = 12	Total, N = 56	Non-O25b/O16, N = 36
Positive virulence traits	n (%)	n (%)	n (%)	n (%)	O25b vs. O16	Non-O25b/O16 vs. O25b/O16 ST131
Less than three	0	0	0	5 (13.8)	0.726	0.119
More or equal to three	44 (100)	12 (100)	56 (100)	31 (86.2)

p-Values ≤0.05 were considered statistically significant.

In comparison between ST131-O25b and ST131-O16 isolates, ST131-O16 isolates exhibited non-significant higher prevalence of six virulence traits and accordingly their aggregate virulence scores were significantly higher compared with the ST131-O25b isolates (mean and range): [5 (3–6) vs. 6 (4–7); p = 0.032].

Discussion

E. coli is the major cause of either community acquired or hospital acquired UTI worldwide. The recently discovered ST131 has markedly contributed to the increased prevalence of antimicrobial resistance among E. coli isolates. Furthermore, scarce data are available regarding this epidemiologically successful clonal group in the Egyptian community, with only one report of its prevalence, which unlike our study, was not performed on fluoroquinolone-resistant E. coli.¹⁹ In this cross-sectional study for the prevalence of the epidemic O25b/O16 ST131 strains of E. coli in community-acquired UTI, about 61% of the fluoroquinolone-resistant E. coli isolates were found to be O25b/O16 ST131. This markedly high frequency of ST131 isolates among fluoroquinolone-resistant E. coli is in accordance with Yokota et al., who found a frequency of 80%.²⁰ This notorious association between fluoroquinolone resistance and ST131 clonal group was previously confirmed by Johnson et al., who found that 96% of their ST131 isolates from UTIs were fluoroquinolone resistant.⁹

The majority of the ST131 isolates in the current study were of the clonal group ST131-O25b (78.5%), while the ST131-O16 subgroup accounted for about 21% of the O25b/O16 ST131 isolates. This is in agreement with a study carried in Saudi Arabia, which showed a similar distribution of both the subgroups.²¹ Olesen et al., also reported this high prevalence of ST131-O25b compared to ST131-O16.²² Notably, this is the first report for the presence of ST131-O16 subgroup in Egypt, which has recently been reported in Japan¹⁴ and Denmark.²² In contrast, the frequency of the O25b clonal group is much higher than that found in a study carried in different countries lying in the Mediterranean area with only a third of their isolates of either O15:K52:H1 or O25:H4 ST131 strains.¹⁰

Due to the marked increase in the fluoroquinolone resistance and its association with ST131, with the increased assumptions that the extensive use of fluoroquinolone has driven the spread of ST131,^2,23 this study focused on studying the prevalence and the characteristics of O25b/O16 ST131 among fluoroquinolone-resistant E. coli. Antibiotic resistance was markedly high in both O25b/O16 ST131 and non-O25b/O16 ST131 isolates with high prevalence of multidrug resistance. Notably, all the ST131 isolates were sensitive to meropenem, providing a better choice for these multiresistant strains. Unexpectedly, the O25b/O16 ST131 isolates were significantly less resistant to amikacin than the non-ST131 isolates. An interesting finding is the significantly higher degree of resistance to extended spectrum cephalosporins in ST131-O25b compared to S131-O16 isolates. This is concurring with Johnson et al., where ST131-O16 isolates showed low resistance levels to ceftriaxone.¹² Moreover, the ST131-O16 showed high resistance to co-trimoxazole, which is in agreement with Matsumura et al.¹⁵

Since the presence, as well as the number, of virulence factors is the predictor of the virulence of E. coli in vivo,²⁴ the virulence score of the isolated E. coli was evaluated. In the current study, all the O25b/O16 ST131 isolates were found to associate with ExPEC status by harboring three or more of the ExPEC virulence factors.^18,22 Moreover, O25b/O16 ST131 isolates showed a significantly higher prevalence of the virulence factors than non-O25b/O16 isolates, which is concurring with the fact of its belonging to the virulent phylogenetic group B2 of E. coli. The previous data contradict what was observed about the low virulence found among ciprofloxacin-resistant E. coli population reported by Kim et al.;²⁵ this controversy could be because the virulent ST131 strains were not searched for in their study unlike our study.

No significant difference was observed between ST131-O25b and ST131-O16 regarding any of the virulence factors; however, ST131-O16 showed a significantly higher virulence score than ST131-O25b. This is in contrast to Matsumura et al.,¹⁵ who found a similar virulence score for O25b and O16 subgroups, which could be due to the difference in the tested virulence factors between this study and theirs.

Finally, a halt for the misuse of fluoroquinolone in the management of UTI would help in preventing the clonal spread of ST131 strains in the Egyptian community.

Conclusion

This is the first report of O16-ST131 in Egypt, which showed a relatively lower frequency than O25b-ST131. The presence of the O25b/O16 ST131 isolates among the community-acquired UTI indicates the endemicity of such emerging multidrug-resistant clone in the community. Implementation of proper urgent interventional measures is required to prevent the spread of the epidemiologically successful ST131 strains.

Footnotes

Disclosure Statement

No competing financial interests exist.

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