Association of Virulent Genes hly, sfa, cnf-1 and pap with Antibiotic Sensitivity in Escherichia coli Strains Isolated from Children with Community-Acquired UTI

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Group: 2010
Subgroup: Volume 12, Issue 1
Date: January 2010
Type: Original Article
Start Page: 33
End Page: 37

Authors:

  • S Farshad
  • Associate Professor of Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Nemazee Hospital, Shiraz, Fars, Iran
  • F Emamghoraishi
  • Pediatrics Department, Jahrom University of Medical Sciences, Jahrom, Fars, Iran
  • A Japoni
  • Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Fars, Iran

      Correspondence:

      Affiliation: Associate Professor of Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Nemazee Hospital
      City, Province: Shiraz, Fars
      Country: Iran
      Tel: +98-711-6474296
      Fax: +98-711-6474303
      E-mail: s_farshad@yahoo.com

Abstract:


Background: Recent studies have suggested that the decrease in the pathogenicity of E. coli is due to acquisition of resistance to some antibiotics. This study was performed to investigate four virulence factors of pap, cnf-1, sfa and hly in resistant E. coli and compare them with susceptible strains of the bacteria isolated from children with community–acquired UTI.

 

Methods: Drug sensitivity of 96 E. coli isolates was evaluated using a disc diffusion method. The prevalence of virulence genes was determined by PCR.

 

Results: E. coli strains showed a high degree of sensitivity to imipenem, amikacin, nitrofurantoin and ciprofloxacin. Approximately, 80.2% of the isolates were resistant to ampicilin. Only 12.5% of the strains were susceptible to all tested antibiotics. PCR showed that cnf-1 (22.9%) was more prevalent than hly (15.6%) and among adhesion coding genes, pap (30.2%) was more prevalent than sfa (18.8%). In all strains, the expression of all virulent genes was less prevalent in most antibiotic resistant groups than in susceptible ones but not statistically significant except for genotypes of pap+-cnf+, pap+- hly+ and cnf+-hly+ with nalidixic acid.

 

Conclusion: We propose that pap and cnf-1 genes in combination with hly gene constitute an uropathogenic genomic configuration which is the characteristic of the nalidixic-acid susceptible E. coli strains, causing urinary tract infection.

  

Keywords: E coli; UTI; pap; hly; sfa; cnf-1; Drug resistance

Manuscript Body:


Introduction

 

Urinary tract infections (UTIs) including cystitis and pyelonephritis are the most common infectious diseases in childhood. Escherichia coli accounts for as much as 90% of the non- community-acquired and 50% of the nosocomial UTIs.1,2 The pathogenic potential of E. coli strains is thought to be dependent on the presence of virulence factors (VFs).3 Urovirulence factors of E. coli analyzed by molecular methods are useful markers for detection of uropathogenic E coli strains.4,5 Some virulence factors such as S fimbriae (sfa), afimbrial adhesion I (afaI), haemolysin (hly), cytotoxic necrotising factor 1 (cnf-1) and aerobactin (aer) play important roles in the pathogenicity of E. coli strains by overcoming host defense mechanisms to cause the disease.6,7 However, most studies showed that in adults, the host defense can prevent adhesion of virulence determinants. These virulence factors are located on large plasmids and/or in particular regions, called "pathogenicity islands" (PAIs), on the chromosome.8 Some research groups have demonstrated that acquisition of resistance may be associated with the loss of virulence factors that might inhibit the invasion of renal and prostatic parenchyma by E. coli.1,9-11 However, this potential reduction in the invasive capacity of resistant E. coli has received little attention in clinical studies. The present study is an attempt to investigate the prevalence of four important virulence factors, cnf-1, sfa, pap and hly, in resistant E. coli and compare them with susceptible strains of the bacteria isolated from urine samples of children with UTI.

 

 

Material and Methods

 

E. coli strains were isolated from urine samples of children aged from l month to 14 years, who presented at Motahary Hospital, Jahrom, Iran. E. coli isolates were identified by standard methods.12 The exclusion criteria were recent antibiotic use during the past 28 days and nosocomial infections which were defined as infections noted 48 hrs post admission or within the 4 weeks following a previous discharge. Positive urine cultures were defined by a growth of single morphotype of colony with counts >105 colony forming unit/ml. Susceptibility of all the isolates to different antibiotics was determined by the disk diffusion method as recommended by the National Committee for Clinical Laboratory Standards,13 with commercial antimicrobial disks (Mast. Co., UK). The antibiotic disks used in this study were ampicilin (10 µg), nalidixic acid (30 µg), cefixime (5 µg), gentamycin (10 µg), nitrofurantoin (300 µg), ciprofloxacin (5 µg), amikacin (30 µg), and imipenem (10 µg). E coli ATCC 25922 was used for quality-control purposes.

The DNA to be amplified was extracted from whole organisms by boiling. The bacteria were harvested from 1.5 ml of an overnight Luria-Bertani broth culture, suspended in sterile distilled water, and incubated at 95oC for 10 min. Following centrifugation of the lysate, the supernatant was stored at -20oC as a template DNA stock. The DNA from Uropathogenic E. coli strain J96 was extracted to be used as a positive control in our PCR reaction.

Detection of pap, sfa, cnf-1 and hly genes was performed by amplifying the genes by PCR. The primers sequences were previously reported,14 and obtained from TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany). Descriptions and sequences of the PCR primers used in this study are displayed in Table 1. Other enzymes and chemicals were provided by Cinnagen Chemical Company (Tehran, Iran). Amplification was performed in a thermal cycler (Eppendorf, Germany) according to the methods described by Yamamoto et al.4 Negative control reactions with distilled water were performed with each batch of amplification to exclude the possibility of any contamination. Expected sizes of the amplicons were ascertained by electrophoresis in 1.5% agarose gel with an appropriate molecular size marker (100-bp DNA ladder, MBI, Fermentas, Lithuania). Statistical analysis was performed using SPSS software for Windows, version 11.5 (SPSS). Chi square or Fisher exact test was used to evaluate the relationship between the variables. A p value less than 0.05 was considered as the significant level.

 

Table 1: PCR primers for amplification of genes hly, cnf-1, pap and sfa sequences in Escherichia  coli strains
isolated from patients with UTI

Gene and DNA region amplified

Primer

Primer Sequence (5/-3/)

Size (bp) of PCR product

hly

F

AACAAGGATAAG CAC TGT TCT GGC T

1,177

R

ACC ATA TAA GCG GTC ATT CCC GTC A 

cnf-1

F

AAG ATG GAG TTT CCT ATG CAG GAG 

498

R

CAT TCA GAG TCC TGC CCT CAT TAT T 

pap

F

GAC GGC TGT ACT GCA GGG TGT GGC G

328

R

ATA TCC TTT CTG CAG GGA TGC AAT A

sfa

F

CTC CGG AGA ACT GGG TGC ATC TTA C

410

R

CAT CAA GCT GTT TGT TCG TCC GCC G

 

 

Results

 

Totally, 96 strains of E. coli were isolated from urine samples of children with UTI, aged 1 month to 14 years (mean 21.8±26.9 months). Drug sensitivity of the isolates was 19.8%, 75.5%, 80.4%, 84.6%, 91.4%, 96.8%, and 96.8% to ampicilin, nalidixic acid, cefixime, gentamycin, ciprofloxacin, nitrofurantoin, and amikacin, respectively (Table2). Sensitivity to imipenem was 100%. Multiple resistances to ampicilin, gentamicin, nalidixic acid and cefixime were seen in 2.1% of the isolates, but no case of multiple drug resistance to all drugs was detected. Only 12.5% of the strains were susceptible to all tested antibiotics. The remaining strains were resistant to one or more antibiotics. Polymerase chain reaction showed that the prevalence of virulent genes ranged from 15.62% for hly to 30.2% for pap. Of toxin coding genes under study, cnf-1 (22.91%) was more prevalent than hly (15.62%). Of adhesion coding genes, pap (30.2%) was more prevalent than sfa (18.75%). Sixty seven (69.8%) strains were negative for the virulent genes.

 

 

Table 2: Antibiotic sensitivity of E. coli strains
isolated from children with UTI

Antibiotic

Sensitive No. (%)

Ampicillin

19 (19.8)

Nalidixic acid

72 (75.5)

Cefixim

77 (80.4)

Gentamycin

81 (84.6)

Ciprofloxacin

88 (91.4)

Nitrifurantoin

93 (96.8)

Amikacin

93 (96.8)

Imipenem

96 (100)

 

As seen in Table 3, the prevalence of all virulent genes was lower for the most antibiotic resistance groups than that for susceptible groups but not statistically significant except for pap and cnf-1 with nalidixic acid (8% and 4.5% positive in resistant vs. 29.4% and 29.6% positive in susceptible groups, respectively, p<0.05). Of the strains positive for these two genes, 94.4% were sensitive and 5.6% were resistant to nalidixic acid. As for the hly, the difference was statistically significant when carried in the strains harboring cnf-1 or pap genes.

 

Table 3: The prevalence of individual virulence factors according to antibiotic resistance status in E. coli strains isolated from children with UTI 

virulence factors 

Antibiotics 

AMP No. (%)* 

GEN No. (%)

NAL No. (%)

NIT No. (%)

AMK No. (%)

CIP No. (%)

CEF No. (%)

IMP No. (%)

pap+

26 (91.3)

  2 (8)

  2 (8)**

0 (0)

0 (0)

1 (3.8)

  3 (12)

0 (0)

pap-

52 (77.4)

12 (18.5)

20 (29.4)

8 (12.1)

3 (4.5)

7 (10.6)

15 (22.7)

1 (1.5)

sfa+

17 (92.3)

  1 (7.1)

  1 (7.1)

0 (0)

0 (0)

0 (0)

  3 (14.3)

0 (0)

sfa-

62 (79.2)

13 (17.1)

21 (26.6)

8 (10.3)

3 (3.8)

8 (10.1)

16 (20.8)

1 (1.3)

cnf-1+

20 (90.5)

  2 (4.8)

  1 (4.5)**

0 (0)

0 (0)

0 (0)

  2 (9.1)

0 (0)

cnf-1-

58 (78.1)

14 (18.8)

22 (29.6)

8 (11.3)

3 (4.2)

8 (11.3)

  7 (23.2)

1 (1.4)

hly+

14 (92.3)

  3 (16.7)

  1 (7.7)

0 (0)

0 (0)

1 (7.7)

  2 (15.4)

0 (0)

hly-

64 (79.2)

12 (15.4)

21 (26.3)

8 (10.1)

3 (3.8)

7 (8.9)

  7 (20.5)

0 (0)

Total

80 (80.2)

15 (15.4)

23 (24.5)

3 (3.2)

3 (3.2)

8 (8.6)

19 (19.6)

1 (1.1)

*Data were shown in number (No.) and (%) of the resistant strains. AMP, ampicilin; GEN, gentamycin; NAL,  nalidixic acid; NIT, nitrofurantoin; AMK, amikacin; CIP, ciprofloxacin; CEF, cefixime, IMP, imipenem; R, resistant; S, sensitive, **, statistically significant.

 

 

Discussion

 

In an attempt to investigate the prevalence of four important virulence factors cnf-1, sfa, pap and hly in resistant in comparison with susceptible uropathogenic E. coli strains isolated from urine samples of children with UTI, we found that pap operon was, as expected, the most prevalent virulence factor identified in the strains. Regarding pap, pooled results with the present data indicate the crucial role of this virulence factor in E. coli-associated UTI.15,16 Moreover, it has recently been shown that the transformation of E. coli with pap sequences is sufficient to convert it to a more potent host response inducer, with P fimbriae lowering the significant bacteriurea threshold.17 The distribution of the sfa operon found among the studied strains was also similar to that in the previously reported data. The prevalence of hly among the collected clinical isolates matches to what reported by other investigators.3,16 However, cnf-1 operon in our study was more prevalent than that found in other studies.3,14,16 This may indicate that cnf-1 gene played an important role in causing UTI in the population under our this study.

Antibiotic sensitivity test showed imipenem as the most effective followed by nitrofurantoin and amikacin in our isolates. All the strains showed the most resistance to ampicillin (Table 2). It is well known that resistance to quinolones such as nalidixic acid is an increasing issue in many parts of the world.18,19 According to recently published data, the rate of quinolone resistant E. coli in the urine has reached 25% which corresponds to our results (24.5%).10 It has also been shown that quinolone resistant uropathogenic E. coli strains (UPEC) express fewer virulence factors than quinolone susceptible strains. Correspondingly, the present data showed that nalidixic-acid-resistant uropathogenic strains were significantly less likely than their susceptible counterparts to harbor pap and cnf-1 operons (p< 0.05). This finding suggests that quinolone resistance could be directly associated with decreased prevalence of virulent genes, as suggested in a previous study.1 Horcajada et al. reported that nalidixic acid resistance was associated with a significantly decreased prevalence of three factors, i.e, sfa, hly and cnf-1.11 It has also been shown by Johnson et al. that the presence of the P fimbriae determinants was also significantly associated with a lack of antimicrobial-agent resistance, as it was the case with the hemolysin determinants.20 In accordance with these data, we also found that all 4 virulent genes under the study were more prevalent in the strains sensitive to nalidixic acid but the sensitivity was significant only for cnf-1 and pap genes. When these two genes were considered along with all other genes, it was revealed that the phenotypes pap+-cnf+, pap+- hly+ and cnf+-hly+ were also significantly less prevalent in strains resistant to nalidixic acid (p<0.05). Depending on the strain, these virulence factors in pathogenic strains are located on chromosomes in one or two PAIs. Meanwhile, it has been found that instability seems to be a characteristic of PAIs in particular pathogens including UPEC.21,22 Middendrof et al. recently reported that the frequency of loss depends on the PAI.23 They also found that among the different environmental conditions such as temperature, osmotic stress, nutrient limiting conditions, or growth in artificial urine, only low temperature and high cell density increased loss of PAI-II536, while the deletion rates of PAI-1 and PAI-V were not affected.23 However, Soto et al. were unable to confirm the spontaneous loss of PAIs during growth in drug-free Luria-Bertani broth.10 On the other hand, Mantel Haenszel’s experiment showed a tendency to a lower prevalence of hly and cnf-1 in nalidixic-acid-resistant E. coli from both cystitis and pyelonephritis than that in their susceptible counterparts. They suggested that clinical syndrome was not a confounding factor.11 According to their data and the results of our study, it seems that the strains lacking some VFs may have lost these virulent genes in exchange for resistance. The role of the SOS system has been analyzed to elucidate the mechanism involved in the induction of the loss of PAIs by quinolones. It is well known that quinolones induce the SOS system response (DNA repairing mechanism), which could favor the splitting of bacteriophages or related sequences such as PAIs from the bacterial chromosome.24 Investigators reported that sub-inhibitory concentrations of quinolones could induce the partial or total in vitro loss of PAIs in UPEC strains. They suggested that this partial or total loss of PAIs induced by quinolones can be observed to occur by a SOS-dependent or independent pathway, respectively.10

According to the studies  on the relationship between urovirulence genes which have copies on chromosome and plasmid, and quinolone sensitivity, it seems that genetic mechanisms related to chromosomes which are involved in producing resistance to quinolone can induce the excision of PAIs genes from chromosome.25 On the other hand, genetic relationship studies such as ribotyping and virulence factors profiling excluded any possibility for acquisition of quinolone resistance by E. coli strains that naturally lack these virulence factors and then spread in a clonal fashion.22 Although all hypotheses almost unanimously have suggested that becoming resistant to antibiotic may affect the absence or presence of these VFs, another explanation for this phenomenon might be that some genetic intermediaries involved in the expression of virulence genes may suppress the expression of the promoter necessary for resistance to quinolones. Although one hypothesis does not exclude the other, further studies are academically warranted to consolidate the findings.

In conclusion, taking into account the findings, we propose that pap and cnf-1 genes in combination with hly gene constitute an uropathogenic genomic configuration which is characteristic of the nalidixic-acid susceptible E. coli strains, causing urinary tract infection. However, more complimentary studies in larger groups of UPEC strains are necessary to confirm these hypotheses.

 

 

Acknowledgments

 

This work was supported by research grant #83-14 from Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences. The authors wish to thank Dr Hassan Khajei for his critical editorial assistance and Mr Mehdi Kalani and Mrs Marzieh Hosseini for their assistance in bacterial isolation, identification, purification and molecular diagnosis.

 

Conflict of interest: None declared.

References: (25)

  1. Vila J, Simon K, Ruiz J, Horcajada JP, Velasco M, Barranco M, Moreno A, Mensa J. Are Quinolone-Resistant Uropathogenic Escherichia coli Less Virulent? J Infect Dis 2002;186:1039-42. [12232848] [doi:10.1086/342955]
  2. Svanborg C, Godaly G. Bacterial virulence in urinary tract infection. Infect Dis Clin North Am 1997;11:513-29. [9378921] [doi:10.1016/S0891-5520(05)70371-8]
  3. Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 1991;4:80-128. [1672263]
  4. Yamamoto S, Terai A, Yuri K, Kurazono H, Takeda Y, Yoshida O. Detection of urovirulence factors in Esherichia coli by multiplex polymerase chain reaction. FEMS Immunol Med Microbiol 1995;12:85-90. [8589667] [doi:10.1111/j.1574-695X.1995.tb00179.x]
  5. Johnson JR, Stapleton AE, Russo TA, Scheutz F, Brown JJ, Maslow JN. Charecteristcs and prevalence within serogroup O4 of a J96-like clonal group of uropathogenic Esherichia coli O4: H5 containing the class I and class III alleles of pap G. Infect Immun 1997;65:2153-9. [9169745]
  6. Donnenberg MS, Welch RA. Virulence determinants of uropathogenic Escherichia coli. in: Mobley HLT, Warren JW. Eds. Urinary Tract Infections: Molecular Pathogenesis and Clinical Management. Washington, DC: ASM Press. 1996; pp. 135-174.
  7. Orskov I, Orskov F. Esherichia coli in extra-intestinal infections. J Hyg (Lond) 1985;95:551-75. [2419401]
  8. Hacker J, Blum-Oehler G, Mühldorfer I, Tschäpe H. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 1997;23:1089-97. [9106201] [doi:10.1046/j.1365-2958.1997.3101672.x]
  9. Velasco M, Horcajada JP, Mensa J, Moreno-Martinez A, Vila J, Martinez JA, Ruiz J, Barranco M, Roig G, Soriano E. Decreased invasive capacity of quinolone-resistant Escherichia coli in patients with urinary tract infections. Clin Infect Dis 2001;33:1682-6. [11595990] [doi:10.1086/323810]
  10. Soto SM, Jimenez de Anta MT, Vila J. Quinolones induce partial or total loss of pathogenicity islands in uropathogenic Escherichia coli by SOS-dependent or -independent pathways, respectively. Antimicrob Agents Chemother 2006;50:649-53. [16436722] [doi:10.1128/AAC.50.2.649-653.2006]
  11. Horcajada JP, Soto S, Gajewski A, Smithson A, Jiménez de Anta MT, Mensa J, Vila J, Johnson JR. Quinolone-resistant uropathogenic Escherichia coli strains from phylogenetic group B2 have fewer virulence factors than their susceptible counterparts. J Clin Microbiol 2005;43: 2962-4. [15956432] [doi:10.1128/JCM.43.6.2962-2964.2005]
  12. Farmer JJ. Enterobacteriaceae: introduction and identification. In: Murray PR, Baron EJ, Phaler MA, Tenover FC, Yolken RH eds: Manual of clinical microbiology. 7nd ed. Washington, ASM Press. 1999; p. 438.
  13. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Eighth informational supplement Villanova, PA: National Committee for Clinical Laboratory Standards (NCCLS) 2000; Approved standard M2 A7.
  14. Arisoy M, Aysev D, Ekim M, Özel D, Köse SK, Özsoy ED, Akar A. Detection of virulence factors of Escherichia coli from children by multiplex polymerase chain reaction. Inter J Clin Pract 2006;60:170-3. [16451289] [doi:10.1111/j.1742-1241.2005.00668.x]
  15. arcia MI, Le Bouguénec C. Role of adhesion in pathogenicity of human uropathogenic and diarrhoeogenic Escherichia coli. Bull Inst Pasteur 1996;94:201-36. [doi:10.1016/S0020-2452(97)86017-4]
  16. Usein CR, Damian M, Tatu-Chitoiu D, Capusa C, Fagaras R, Tudorache D, Nica M, Le Bouguénec C. Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases. J Cell Mol Med 2001;5:303-10. [12067489] [doi:10.1111/j.1582-4934.2001.tb00164.x]
  17. Wullt B. The role of P fimbriae for Escherichia coli establishment and mucosal inflammation in the human urinary tract. Int J Antimicrob Agents 2003;21:605-21. [13678032] [doi:10.1016/S0924-8579(02)00328-X]
  18. Martínez-Martínez L, Fernández F, Perea EJ. Relationship between haemolysis production and resistance to fluoroquinolones among clinical isolates of Escherichia coli. J Antimicrob Chemother 1999;43:277-9. [11252335] [doi:10.1093/jac/43.2.277]
  19. Lepelletier D, Caroff N, Reynaud A, Richet H. Escherichia coli: epidemiology and analysis of risk factors for infections caused by resistant strains. Clin Infect Dis 1999;29:548-52. [10530446] [doi:10.1086/598632]
  20. Johnson JR, Moseley SL, Roberts PL, Stamm WE. Aerobactin and other virulence factor genes among strains of Escherichia coli causing urosepsis: association with patient characteristics. Infect Immun 1988;56:405-12. [2892793]
  21. Blum G, Ott M, Lischewski A, Ritter A, Imrich H, Tschäpe H, Haker J. Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen. Infect Immun 1994;62:606-14. [7507897]
  22. Ott M. Dynamics of the bacterial genome: deletions and integrations as mechanisms of bacterial virulence determination. Zentbl Bakteriol 1993;278:457-68. [8353320]
  23. Middendorf B, Hochhut B, Leipold K, Dobrindt U, Blum-Oehler G, Hacker J. Instability of pathogenicity islands in uropathogenic Escherichia coli 536. J Bacteriol 2004;186:3086-96. [15126470] [doi:10.1128/JB.186.10.3086-3096.2004]
  24. Phillips I, Culebras E, Moreno F, Baquero F. Induction of the SOS response by new 4-quinolones. J Antimicrob Chemother 1987;20:631-8. [3323160] [doi:10.1093/jac/20.5.631]
  25. Bagel S, Hüllen V, Wiedemann B, Heisig P. Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrob Agents Chemother 1999;43:868-75. [10103193]