Clonal Dissemination of Genetically Diverse Fluoroquinolone-Resistant Extended-Spectrum Beta-Lactamase ( ESBL )-Producing Escherichia coli ST 131 in a Veterans Hospital in Southern Taiwan

Uropathogenic Escherichia coli is the common pathogen to cause urinary tract infections (UTIs) and have become multidrug-resistant (MDR) extended-spectrum β-lactamase (ESBL) producers. The differences in the antimicrobial susceptibility, 5 bla genes, 12 virulence genes of 87 clinical ESBL-producing E. coli isolates and genomic variations and sequence types of 18 recurrent and repeated isolates from 9 patients were investigated. The 87 MDR-ESBL isolates collected mainly from indwelling urinary catheters (IUCs) and UTIs were highly resistant to fluoroquinolones, with over 50% of the isolates being resistant to cefepime and piperacillin/tazobactam and a few being resistant to carbapenem. These isolates carried at least two of the five bla genes examined, with the highest prevalence (87.4%) found for blaCTX-M (blaCTX-M3-like and blaCTX-M14-like), followed by blaCMY-2 (80.5%) and blaSHV (56.3%). The predominant virulence genes were the fimbriae gene fimH and the toxin genes cnf1 and hlyA in blood isolates and the capsule gene kpsMTII in UTI and blood isolates. Over 80% of the isolates carried yersiniabactin and aerobactin of siderophores. In 18 Contributed equally Corresponding author.

Genetically related strains with multidrug resistance are the characteristics of UTI-and catheter-associated UTIs (CAUTIs)-associated E. coli, which carry similar virulence genes as UPEC [7] and account for more than 40% of all nosocomial infections in hospitals and nursing homes [3] [7]- [9].Important factors that lead to ESBL-producing E. coli-associated nosocomial infection are the use of extended-spectrum cephalosporins and urethral catheterization [10]- [12].The most prevalent uropathogenic E. coli ST131 strains differ in their uropathogenic virulence factors, are associated with phylogroup B2, and carry IncF, IncN, IncA/C, and IncI1 plasmids, with a size ranging from 50 to >200 kb [13]- [16].The aims of this study were to characterize the ESBL-producing E. coli with differences in uropathogenic virulence genes and to investigate the genomic and plasmid variations of E. coli ST131 based on nine episodes of recurrent infections and repeated isolation.

Bacterial Sources
A total of 101 clinical E. coli isolates were collected consecutively from IUCs, UTIs, blood, sputum, wounds and ears of patients from August 2010 to February 2011 and were identified as ESBL producers in the Medical Laboratory Division of Chiayi Branch, Taichung Veterans General Hospital.After the further identification of E. coli by biochemical methods, confirmation of ESBL at National Chiayi University and the removal of genetically identical isolates from same patient, 87 ESBL-producing isolates were analyzed.All of the E. coli isolates were routinely incubated on EMB agar for further use.Furthermore, 18 isolates from 9 patients who were infected recurrently or at different sites within a short period were investigated.A recurrent E. coli infection was defined as a patient who was infected by a culture-proven E. coli more than 1 month after the completion of the initial antimicrobial therapy.

PCR Detection of Genes for Antimicrobial Resistance and Virulence Factors
The primers used to amplify genes for five β-lactamases and 12 virulence factors are listed in Table 1.Three multiplex PCR sets were developed by modifying previous primer sequences.Antimicrobial resistance genes were detected by Multiplex PCR I for bla SHV, bla CTX-M3-like , bla CTX-M14-like , bla TEM and bla DHA and by single PCR for bla CMY-2 .Twelve virulence genes were amplified by two multiplex PCR sets: hlyA, usp, sat, fyuA, ironN, iutA and iucD for multiplex PCR II and fimH, cnf1, traT, kpsMTII and papGII for Multiplex PCR III.All PCR products were separated by electrophoresis on a 2% agarose gel at 50 V for 120 min.After ethidium bromide staining and UV illumination, the gel image was recorded and analyzed.

Genomic, Plasmid, Multilocus Sequence Typing (MLST), and Sequence Analysis of Recurrent Isolates
The procedure for PFGE was described previously [18].Briefly, 10 U of XbaI were used for the restriction digestion.Isolates were defined as the subgenotype for those with ≤3 fragment differences and as the genotype for those with >3 fragment differences.Plasmid size and number were analyzed using the Kado-Liu method [19], and Salmonella Choleraesuis OU7085 (6.6-and 50-kb plasmids) and S. Choleraesuis OU7526 (50-and 90-kb plasmids) as a control.Furthermore, the plasmid replicon groups FIA, FIB, FIC, HI1, HI2, I1-Igamma, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA were identified for each isolate by PCR amplification [20].Full-length bla CTX-M-3-like was sequenced and aligned to the NCBI database.MLST was performed using previously described methods [21].After PCR amplification and sequencing, MLST ST types were determined at http://mlst.ucc.ie/.

Plasmid Conjugation
Sodium azide-susceptible clinical recurrent E. coli isolates and sodium azide-resistant E. coli strain J53 were used as donors and recipient, respectively, for conjugation experiments.After the co-culture of recipient and donor strains on filter paper on MHA agar overnight, the mixture was plated onto MacConkey agar with the appropriate antibiotic and sodium azide.Plasmid profile of the transconjugants was analyzed using the Kado-Liu method.The replicons of the conjugation plasmid were examined as above [20].

Antimicrobial Resistance
In general, all isolates were multidrug resistant to AMP, CRO and CTX and PIP with the least antimicrobial resistance for the urine group and the highest antimicrobial resistance for the IUC group (Table 2).The average resistance rate was 54% to TZP (range: 42.3% for the urine group to 66.7% for the wound group); 76% to CAZ (range: 65.4% in the urine group to 91.7% in the blood group); 60.9% to FEP (range: 57.7% for the urine group to 70% for the sputum group); less than 10% to carbapenems, including ETP, IPM and MEM, with the value observed highest in the urine group; near 90% to fluoroquinolones; over 95% to nalidixic acid; and near 90% to CIP, LVX, and MXF, particularly differing between the urine and IUC groups, with the lowest found in the blood and wound groups.

ESBL-Related bla Genes
The bla genes differed in prevalence among the isolation source (Table 3).The most prevalent bla gene was bla CTX-M (87.4%; 54.0% for the bla CTX-M3-like gene and 33.3% for bla CTX-M14-like ), followed by bla CMY-2 (80.5%),  bla SHV (56.3%), bla TEM (36.8%) and bla DHA (2.3%).Overall, 67.4% of the isolates carried at least 3 bla genes.For example, bla CTX-M-3-like , bla CMY , and bla SHV were observed in 31.6% of the isolates.The prevalence of bla TEM ranged from 16.7% for the wound group to 53.8% in the urine group; the prevalence of bla CMY-2 ranged from 70.0% for the sputum group to 85.4% for the IUC group.The prevalence of bla SHV ranged from 50% for the blood and sputum group to 66.7% in the wound group.The prevalence of bla CTX-M3-like ranged from 52% for the urine group and blood group to 66.7% for the wound group.The prevalence of bla CTX-M14-like ranged from 28.1% in the IUC group to 42.3% in the urine group.The bla CTX-M3-like and bla CTX-M14-like that were present were mutually exclusive.The gene bla DHA was identified in only two urine isolates.

Differences in Virulence Genes in Pathogenicity Islands (PAI 536 ) of UPEC
In contrast to the more than 80% of isolates that carried type I fimbrae fimH, the prevalence of the pathogenicity island-associated fimbrial gene papGII ranged from 22% for the wound group to 67% for the blood group (Table 3).Over 50% of the urine and bacteremia isolates carried the capsular gene kpsMTII.The toxin genes cnf1 and hlyA were more highly observed in bacteremia isolates.Among the three siderophore types, aerobactin genes (iutA and iucD) and the yersiniabactin gene (fyuA) were present in over 80% of the isolates, whereas the salmochelin gene iroN was nearly absent.The fyuA gene was present in over 90% of the wound and urine groups and in only 56% of the sputum group.The uropathogenic protein gene usp was found in more bacteremia and UTI groups (75% and 68%) than sputum group (55%).The gene sat was most often found in the IUC group (Table 4).

Genetic Variations in 18 E. coli Isolates from Recurrent Infections and Different Locations in Same Patient
These isolates were obtained mostly from urine and IUC and belonged to uropathogenic ST131 of pulsotypes I (IA and IB) and II (IIA and IIB), except ST95 of pulsotype III for patient 5 (Table 5).Furthermore, all pulsotype IA isolates were collected from ward 22W.Among the virulence genes, these isolates carried the siderophore genes iutAB and fyuA and usp (Table 5).Except for isolate 9, all isolates missed at least one virulence gene from fimH, papGII, kpsMTII, cnf1, and hlyA.The ST 95 isolates from patient 5 carried fewer siderophore genes, lacked the genes traT and sat and were sensitive to fluoroquinolone compared to the ST131 isolates, which were all fluoroquinolone-resistant and resistant to cefotaxime (CTX), ceftazidime (CAZ), and ceftriaxone (CRO), of the third-generation cephalosporins.Furthermore, few isolates were resistant to fourth-generation cefepime (FEF).Among the four bla genes examined by PCR and sequencing, bla CTM-M15 was the most prevalent bla gene and was potentially located on the IncF plasmid (Figure 1).Furthermore, the AmpC gene bla CMY-2 was detected with high prevalence (15/18).Plasmid profile differed in size and number, ranging from 6.6 kb to larger than 90 kb (Figure 1).Four plasmids were common, with sizes less than 50-kb (1), 50 -90 kb (2) and larger than 90 kb (1).Among the 18 plasmid replicon types tested, only five replicon types A/C, I1, FIA, FIB and Y were identified, and the plasmid with these replicons could be conjugated (Table 5).Replicon types combined with PFGE, ST, and plasmid analysis demonstrated identical isolates in patients 2, 5, 6, 7, and 8 and different isolates in patients 1, 3, 4, and 9.

Discussion
E. coli is the major pathogen for clonal community-acquired and nosocomial UTIs [1] [22]- [24].In addition, CAUTIs are a major nosocomial infection in hospitals and nursing homes [8].In this study, in a nursing home in this hospital, CAUTIs (IUCs) were more common than UTIs (Table 2).We confirmed that fluoroquinoloneresistant ESBL-producing E. coli infections caused more than 80% of the UTIs and bacteremia observed in older patients [25].The pathogenicity island (PAI) I 536 -IV 536 carries genes for UPEC to cause bacteremia [6].Indeed, virulence genes of PAI-I 536 -IV 536 were found to be more common in the isolates from urine, IUC and blood than in those from sputum and wounds, but these genes differed among the UTI, IUC and blood groups.For example, papGII and kpsMTII were more common in the blood group than in IUCs and UTIs, and the fimbriae genes fimH and papGII, toxin genes cnf1, hlyA, and sat were more common in the IUC group than in the UTI group (Table 3).These results suggest the different origins of blood, IUC and urine groups.Our study also demonstrated that the ESBL-producing isolates and recurrent UPEC ST131 mostly consisted of yersiniabactin and aerobactin genes, and lacked the salmochelin gene (Tables 3-5), which confirmed that yersiniabactin and aerobactin are more prevalent in relapsed UPEC isolates [26].Additionally, yersiniabactin are more common in ST131 than non-ST131 strains [27], implying different strains between sputum isolates and the blood and UTI isolates (Table 3).
E. coli ST131 and ST95 isolates have been the major UPEC isolates causing UTI in Canada and the USA [28] [29].Our study demonstrated clonal dissemination of ST131 with bla CTX-M-15 , such as pulsotype IA isolates in ward 22W and most pulsotype IB isolates in 21W and SCU (Table 5, Figure 1), which confirmed that the clonal dissemination of UPEC infection is often responsible for recurrent infection in patients due to ineffective treatment [26] as well as for recurrent bloodstream infection in elderly patients [30].In addition, these isolates frequently carry the CTX-M ESBL enzyme [31].
Fluoroquinolone-resistant E. coli ST131 carried a plasmid that was capable of conjugation in which replicon types IncF were the most prevalent.Inc I1-Iγ, K, HI2 and B/O [32] and E. coli ST131 were isolated in UK river sediment and carried bla CTX-M-15 , which is associated with the replicon IncF plasmid [33].Furthermore, bla CTX-M-15 was identified in 100-335-kb Inc FII, IncHI2, IncY plasmids and other non-typeable plasmids, with a size ranging from 70 -330 kb [34].Carbapenem-resistant E. coli ST131with bla KPC on the conjugable plasmid has been identified [35].However, we only observed reduced susceptibility to carbapenem in this study.In the present study, fluoroquinolone-resistant ESBL-producing E. coli ST131 with bla CTX-M-15 evolved differently via an increase in genomic variation (Figure 1) and the transfer of plasmids that differed in size, number and replicon types, suggesting a genome plasticity in both the plasmid and chromosome that have resulted in the greater pathogenicity of ST131 and its rapid dissemination worldwide.

Conclusion
In conclusion, fluoroquinolone-resistant ESBL-producing E. coli differed in the virulence genes of PAI-I 536 -IV 536 of UPEC.Uropathogenic E. coli ST131 was dominant in this hospital, and it evolves genetically via plasmid transfer and chromosome variations.

Figure 1 .
Figure 1.Plasmid profile and replicon type and bla genes in recurrent E. coli isolates.A: Plasmid analysis.Size standards are 90-kb and 50-kb plasmids of OU7526 and the 50-kb and 6.6-kbplasmids of OU7085.

Table 1 .
Primer sequences and PCR product size of genes for antimicrobial resistance and virulence factors.

Table 3 .
Prevalence of bla genes in ESBL-producing E. coli isolates from different sources.