Escherichia Coli: Characteristics of Carbapenem Resistance and Virulence Factors

ABSTRACT

In this study, fifty Escherichia coli strains were analyzed by multiplex polymerase chain reaction for the genes expressed carbapenemase and virulence factors in order to determine the presence of carbapenemase and nine virulence factors and investigate the association between these two characteristics. When carbapenemase susceptibility was taken into consideration, OXA-48 type carbapenemase was determined for 22% of the total strains. Also, the frequency of virulence gene regions in E.coli infections and virulence gene profiles of these isolates were examined and the frequency of pap, afa, sfa, fimA, iroN, aer, iutA, hly and cnf-1 genes were 24, 38, 20, 84, 28, 90, 92, 10 and 34% respectively. A significant correlation was found between the presence of fimA and afa gene regions and carbapenem susceptibility (P< 0.05). Based on the combination of carbapenemase and virulence factor genes, 24 different gene profiles were determined for all strains. The results of the study appear to indicate that fimA and afa genes correlate with carbapenem susceptibility, the relations of fimA with urinary tract infections and pap with complicated urinary tract infections. It also indicates that sfa and afa genes correlate with other infections except urinary tract infections.

Key words: Carbapenemase; Escherichia coli; multiplex polymerase chain reaction; virulence factor.

INTRODUCTION

Escherichia coli causes urinary tract infections, neonatal meningitis, sepsis and intestinal infections more frequently than other members of the Enterobacteriaceae and it is responsible for 80% of community-acquired urinary tract infections.¹

Extended-spectrum beta-lactamases (ESBL) which hydrolyze penicillins, cephalosporins, monobactams and generally inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam can be produced by the members of Enterobacteriaceae.² The increasing usage of carbapenems, the first treatment option in ESBL producing E. coli infections, has brought out the problem of carbapenem resistance.³ Carbapenems was firstly identified in clinical isolate of Enterobacter cloacae⁴ and then carbapenem-resistant Enterobacteriaceae was reported from around the world.⁵ Serine carbapenemases (KPC and OXA-48) and metallo-beta-lactamases (VIM, IMP and NDM) are the most commonly seen among carbapenemases.⁵

E.coli strains have some gene regions responsible for virulence factors which may encode adhesins, toxins, siderophores and haemolysin. Type 1 fimbriae, coded by plasmid-mediated fimA gene and commonly found in these strains from lower urinary system infections, enable E.coli to adhere to human ureteral mucosa epithelial cells.⁶ P fimbriae that is expressed by plasmid-mediated pap (pyelonephritis-associated pili) gene, is produced by E.coli colonization of upper urinary system.⁷ Afimbrial adhesin encoded by plasmid- or chromosom- mediated afa and S fimbriae encoded by plasmid sfa gene regions are commonly found in urinary system infection originated in isolates as well as sepsis and meningitis.^8,9

Siderophores commonly found in E.coli isolates are aerobactin and ferric aerobactin, encoded by aer and iutA genes.¹⁰ Also, salmochelin type siderophore encoded by iroN gene in recent years has become important.¹¹ These genes are expressed as plasmid-mediated and they are especially related to necrotoxigenic, uropathogenic and septicemic E.coli isolates causing urinary tract infections, septicemia, bacteremia and systemic infections. ^6,12 Also, cytotoxic necrotizing factor 1 (cnf-1) and haemolysin (hly) toxins are important for diversity of E.coli infections^13,14and these gene regions expressed by plasmids are mostly produced by uropathogenic E.coli strains.¹⁵

The aim of the study is to determine the presence of carbapenemase and virulence factors of E.coli clinical specimens and evaluate the possible correlations between carbapenem resistance and virulence factors. Also, the frequency of virulence gene regions in E.coli infections and virulence genotypes of these isolates were determined.

MATERIAL AND METHODS

Bacterial strains and identification

In this study, E.coli strains (n=50), obtained from clinical specimens including abscess, bronchial, urea, blood, plevral efusion, tracheal, wound, catheter, body and ear fluid, were collected from three different hospitals in Ankara. Conventional methodology (Gram staining, hemolysis of blood agar, string test, IMViC tests, lactose fermentation, ornithine decarboxylase and motility tests), automatized system (Vitek-32 System, bioMerrieux, France) and CHROMagar Orientation (CHROMagar Company, Paris, France) were used for identification of the strains.

Antimicrobial susceptibility testing

Susceptibility to extended spectrum beta lactamases (ESBL) and carbapenems were determined with CHROMagar ESBL (CHROMagar Company, Paris, France)¹⁶ and CHROMagar KPC (CHROMagar Company, Paris, France)¹⁷ respectively.

DNA isolation

Genomic and plasmid DNA were isolated using NucleoSpin(r)Tissue (Macherey-Nagel, Germany) and NucleoSpin(r)Plasmid (Macherey-Nagel, Germany) and were stored in -20ºC.

Multiplex PCR analysis of carbapenemase gene regions

The reaction conditions were modified from Poirel et al. ¹⁸. The Multiplex PCR mixture consisted of 1XPCR buffer (20mM Tris HCl, 10mM (NH₄)₂SO₄, 10mM KCl, 2mM MgSO₄, 0,1% Triton X-100), 0,05mM dNTP, 2U Taq polymerase (NEB, Beverly, MA), 50 μmol/L each of primers (NEB, Beverly, MA) and 2μl DNA. The primers used in this study are listed in Table 1.

Table 1
Carbapenemase gene regions¹⁸

Primer	Sequence* (5'-3')	Gene	Product size (bp)
KPC-F	CGTCTAGTTCTGCTGTCTTG	blaKPC	798
KPC-R	CTTGTCATCCTTGTTAGGCG
NDM-1-F	GGTTTGGCGATCTGGTTTTC	blaNDM-1	621
NDM-1-R	CGGAATGGCTCATCACGATC
OXA-48-F	GCGTGGTTAAGGATGAACAC	blaOXA-48	438
OXA-48-R	CATCAAGTTCAACCCAACCG
IMP-F	GGAATAGAGTGGCTTAAYTCTC	blaIMP	232
IMP-R	GGTTTAAYAAAACAACCACC
VIM-F	GATGGTGTTTGGTCGCATA	blaVIM	390
VIM-R	CGAATGCGCAGCACCAG

• *Y=C or T
• IMP, imipenem metallo-β-lactamase; KPC, Klebsiella pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; OXA, oxacillinase; VIM, verona integron-encoded metallo-β-lactamase.

Amplification was carried out with the following thermal cycling conditions: 5 minutes of pre-denaturation at 95°C, followed by 35 cycles: 1 minute at 95°C, 1 minute at 52°C, 1 minute at 72°C and 10 minutes of final elongation at 72°C (Sensoquest Labcycler, Germany).

Multiplex PCR analysis of virulence gene regions

The mix for the detection of pap genes consisted of 2XPCR buffer (40mM Tris HCl, 20mM (NH₄)₂SO₄, 20mM KCl, 4mM MgSO₄, 0,2% Triton X-100), 0,2mM dNTP, 2U Taq polymerase (NEB, Beverly, MA), 2,5 μmol/L each of primers (NEB, Beverly, MA) and 2,5μl DNA. The mix for the detection of other groups (afa-sfa-fimA, hly-iroN-aer-cnf1-iutA) was at the same concentrations. The primers used in this study are listed in Table 2.

Table 2
Virulence gene regions of E.coli ^13,14,19

Primer	Sequence (5'-3')	Product size (bp)
fimA		447
fimA-F	GTTGTTCTGTCGGCTCTGTC
fimA-R	ATGGTGTTGGTTCCGTTATTC
pap
pap1	GACGGCTGTACTGCAGGGTGTGGCG	328
pap2	ATATCCTTTCTGCAGGGATGCAATA
pap3	GCAACAGCAACGCTGGTTGCATCAT	336
pap4	AGAGAGAGCCACTCTTATACGGACA
sfa		410
sfa-F	CTCCGGAGAACTGGGTGCATCTTAC
sfa-R	CGGAGGAGTAATTACAAACCTGGCA
aer
aer-F	TACCGGATTGTCATATGCAGACCG	602
aer-R	AATATCTTCCTCCAGTCCGGAGAAG
cnf-1
cnf-F	AAGATGGAGTTTCCTATGCAGGAG	498
cnf-R	CATTCAGAGTCCTGCCCTCATTATT
hly
hly-F	AACAAGGATAAGCACTGTTCTGGCT	1177
hly-R	ACCATATAAGCGGTCATTCCCGTCA
afa
afa-F	GCTGGGCAGCAAACTGATAACTCTC	750
afa-R	CATCAAGCTGTTTGTTCGTCCGCCG
iutA
iutA-F	GGCTGGACATCATGGGAACTGG	300
iutA-R	CGTCGGGAACGGGTAGAATCG
iroN
iroN-F	AAGTCAAAGCAGGGGTTGCCCG	665
iroN-R	GACGCCGACATTAAGACGCAG

Amplification was carried out with the following thermal cycling conditions: 5 minutes of pre-denaturation at 95°C, fol lowed by 30 cycles: 1 minute at 94°C, 1 min ute at 58°C, 1 minute at 72°C and 10 minutes of final elongation at 72°C (Sensoquest Labcy cler, Germany).

PCR products were analyzed by electrophoresis in a 1.8% agarose gel at 150 V for 2 h in 1 × TBE (89 mM Tris, 89 mM Boric Acid and 2 mM EDTA) containing 0.05 mg/L ethidium bromide and images were captured by Gel Logic 200 Molecular Imaging System (Kodak; Rochester).

Data analysis

The Fisher's Exact Test were performed for all clinical data. P value of <0.05 was considered statistically significant.

RESULTS

In total, 50 Escherichia coli strains were identified by microbiology standard methods and chromogenic medium.

Analysis of the carbapenemase gene regions

Among E.coli isolates, plasmid mediated oxacillinase (OXA-48) gene was determined in 11 strains (22%) (Fig. 1). Carbapenem gene regions of these isolates are given in Figure 1. Similarly, car bapenem resistance in these strains was determined phenotypically, using CHROMagar KPC.

Figure 1
OXA-48 type carbapenemase of E.coli strains (28-41; E.coli, NC; Negative control, M; 100 bp DNA molecular marker).

Analysis of the virulence gene regions

In this study, nine virulence gene regions (pap, afa, fimA, sfa, hly, iroN, aer, cnf-1 and iutA) for E.coli isolates were analyzed by Multiplex PCR. These gene regions are shown in Figure 2.

Figure 2
Determined virulence gene regions in E.coli (pap; 328-336 bp, afa; 750 bp, fimA; 447 bp, sfa; 410 bp, hly; 1177 bp, iroN; 665 bp, aer; 602 bp, cnf-1; 498 bp, iutA; 300 bp).

The distribution of the virulence gene regions in E.coli strains are shown in Figure 3. A high prevalence of virulence genes expressing siderophores (iutA; 92%, aer; 90%) in E.coli strains was observed. The Multiplex PCR results showed that among the strains, 84% were positive for fimA, 38 for afa, 34 for cnf-1, 28 for iroN, 24 for pap, 20 for afa and 10 for hly. The least common virulence gene region among strains is hly gene region responsible for hemolysis.

Figure 3
Distribution of virulence gene regions in E.coli strains. "n" is the number of isolates that were found to possess a given gene; "%" represents n as the percentage of the 50 strains studied.

Distribution (%) of virulence factors in different clinical E.coli strains, as distribution of gene regions related with fimbria types (afa, fimA, sfa and pap), siderophore formation (iroN, iutA and aer) and other virulence factors (cnf-1 and hly), are shown in Figure 4. Great diversity of virulence in urine isolates were observed, whereas any virulence gene is not found in catheter.

Figure 4
Distribution (%) of virulence genes (a; fimbriae, b; siderophore, c; others) in different clinical sources (urine; 35, wound; 6, blood; 2, bronchial; 1, ear fluid; 1, body fluid; 1, abscess; 1, catheter; 1, plevral efusion; 1, tracheal; 1 strain).

Virulence factor gene profiles of E. coli isolates

In this study, when the carbapenem and virulence gene profiles are evaluated and 24 types of virulence profiles are identified (Table 3). In total, 98% of the strains carry at least one virulence gene.

Table 3
Carbapenem resistance and virulence gene profiles of E.coli strains

Strains	Clinical source	Carbapenem resistance	Virulence gene profiles
29	Catheter	OXA-48	not determined
44	Body fluid	susceptible	fimA
48	Urine	susceptible
41	Urine	OXA-48	fimA, iutA
1	Abscess	susceptible	fimA, aer
28	Plevral efusion	OXA-48	aer, iutA
32	Wound	OXA-48
35	Wound	OXA-48
36	Tracheal	OXA-48
30	Urine	susceptible
49	Urine	susceptible
27	Urine	susceptible	aer, iutA, cnf-1
7	Urine	susceptible	fimA, aer, iutA
19	Urine	susceptible
40	Urine	susceptible
43	Wound	susceptible
33	Urine	OXA-48	fimA, sfa, aer, iutA
18	Urine	susceptible	fimA, pap, aer, iutA
45	Urine	susceptible
46	Ear fluid	susceptible
37	Blood	OXA-48	fimA, afa, aer, iutA
5	Urine	susceptible
8	Urine	susceptible
9	Urine	susceptible
24	Wound	susceptible
25	Wound	susceptible
50	Urine	susceptible
31	Urine	OXA-48	fimA, aer, iutA, cnf-1
4	Urine	susceptible
6	Urine	susceptible
17	Blood	susceptible
34	Urine	susceptible	fimA, sfa, aer, iutA, iroN
38	Urine	OXA-48	fimA, pap, aer, iutA, hly
16	Urine	susceptible	fimA, pap, aer, iutA, iroN
21	Urine	susceptible	fimA, pap, afa, aer, iutA
26	Urine	susceptible
3	Urine	susceptible	fimA, afa, aer, iutA, cnf-1
20	Urine	susceptible
12	Urine	susceptible	fimA, pap, afa, aer, iutA, iroN
15	Urine	susceptible
23	Urine	susceptible	fimA, sfa, afa, iutA, iroN, cnf-1
2	Bronchial	susceptible	fimA, afa, aer, iutA, iroN, cnf-1
13	Urine	susceptible
22	Urine	susceptible	fimA, sfa, aer, iutA, iroN, hly, cnf-1
47	Urine	susceptible
11	Urine	susceptible	fimA, sfa, afa, aer, iutA, iroN, cnf-1
42	Wound	susceptible	fimA, pap, sfa, aer, iutA, iroN, cnf-1
39	Urine	OXA-48	fimA, pap, sfa, aer, iutA, iroN, hly, cnf-1
10	Urine	susceptible	fimA, sfa, afa, aer, iutA, iroN, hly, cnf-1
14	Urine	susceptible	fimA, pap, sfa, afa, aer, iutA, iroN, cnf-1

When the virulence profiles in Table 3 are examined, carbapenem resistant E.coli isolated from catheter (strain 29) was not included any virulence gene regions. On the other hand, carbapenem resistant E.coli (strain 39) isolated from urine has eight virulence gene regions and the strain is the most virulent isolate among other.

Correlation between virulence genes and carbapenem resistance

The overall virulence factor productions between carbapenem resistant (n=11) and carbapenem susceptible (n= 39) E.coli strains are shown in Table 4. Correlation between the presence of the fimA or afa gene regions and carbapenem resistance was statistically significant (P< 0.05). Most of E.coli strains carrying these genes were susceptible to carbapenem.

Table 4
Distribution of carbapenem resistant and susceptible E.coli strains

Virulence factors	Carbapenem resistant strains (n=11) (22%)	Carbapenem susceptible strains (n=39) (78%)	P value
pap	2 (18)	10 (26)	>0.05
sfa	2 (18)	8 (21)	>0.05
fimA	6 (55)	36 (92)	<0.05
afa	1 (9)	18 (46)	<0.05
hly	2 (18)	3 (8)	>0.05
cnf-1	2 (18)	15 (38)	>0.05
iutA	10 (91)	36 (92)	>0.05
aer	9 (82)	36 (92)	>0.05
iroN	1 (9)	13 (33)	>0.05

DISCUSSION

Serine carbapenemases (KPC and OXA-48) and metallo-beta-lactamases (VIM, IMP and NDM) are frequently found in the members of the Enterobacteriaceae. ²⁰ OXA-48 type carbapenemases are commonly reported in E.coli²¹ and recently NDM²², IMP²³ and KPC²⁴ type carbapenemases have also been determined. In our study, only OXA-48 type resistance is found in E.coli isolates. This plasmid-based resistance has been reported in Turkey and around the world and it is more common than other types of resistance.^5,25,26

Siderophores are the most important virulence factor supporting bacterial infection in tissues and blood, in case of there is iron deficiency. Aerobactin and ferric aerobactin expressed by aer and iutA gene regions are important siderophores in E.coli virulence and commonly found in many isolates.^10,11 In this study, it is found that the most common regions for these genes were related with siderophores.

aer gene was reported to have the highest ratio among the virulence factor genes.^13,27 Besides, a study with extrainstestinal E.coli isolates, the highest iutA gene region within virulence gene regions was found and this gene region was not determined in the control isolates.¹² In our study, another siderophore related gene region is iroN. This gene region is responsible of salmochelin and causes invasion in urothelial cells.¹¹ And also, this gene region was reported to have the highest ratio after iutA.¹²

Another virulence gene region found with high incidence in our study was fimA which is related to Type I fimbriae. This gene, commonly found most of the E.coli isolates, is observed both pathogenic and commensal isolates despite being an important virulence factor.^6,8 Similarly, it was reported in many studies that this gene region was more than other fimbria types.^12,27,28

Urinary tract infections (cystitis, pyelonephritis etc.) which are considerably common and seen by many different symptoms^8,29, the virulence factor diversity is mostly found in urine samples in this study. The most important virulence factors for urinary tract infections are fimbriae. It was reported that no important difference in presence frequency of type I fimbria between low and high virulence isolates in the urinary tract.³⁰ In our study, fimA was found in most of the urine samples. Similarly, in Johnson et al., this gene region, despite commonly found in most E.coli isolates, is thought to be specific gene region for urinary tract infection. ²⁸

pap gene region, particularly with pyelonephritis, plays an important role in pathogenesis of uropathogenic E.coli, and this gene region is highly found in the isolates of urinary tract infection²⁹ and kidneys.³¹ Thus, this gene region is found almost three-fold lower than the results reported before. On the other hand similar result were reported in our study.¹³ For all the studies, even for the ones with high or low percentage, it is clear that this gene region is mostly related to complicated urinary tract infections.

The decay acceleration factor (DEF) in humans, which is a glycoprotein found in hemopoietic, endothelial, intestinal and urinary cells, is a receptor for afimbrial adhesin expressed with afa gene region.⁶ In our study, this gene was found in urine samples as well as blood and bronchial samples. Sfa gene region related with S fimbria is found in urinary tract infections as well as infections such as sepsis, meningitis like afa gene region.⁸ These gene regions were reported in none²⁹ or low percentage²⁸ of the samples isolated complicated urinary tract infection and there are no specific gene regions for these infections.

Siderophores in urinary tract infections are virulence factors frequently found in most pathogenic types of E.coli after fimbria. Even though, iutA gene region was reported in high incidence in complicated urinary tract infection compared to control group and acute cholangite.²⁹ Landgraf et al. reported that relation between iutA gene and uropathogenics was low.³² Also, salmochelin (iroN) was found higher than ferric aerobactin (iutA) in several types' urinary tract infections.^11,28 In our study, presence of aer and iutA gene regions in many types of clinical strains and iroN gene in urine shows that this gene is a siderophore specific to urinary tract infection.

Hemolysin (hly) is produced by various pathogenic types of E.coli causing extraintestinal and intestinal infections, but its effect on virulence is not completely clarified.^6,8hly gen region being related with complicated urinary tract infections such as pyelonephritis and cystitis is reported by many researchers.^8,27,29 Similarly, cnf-1 gene region is produced in one third of the isolates causing pyelonephritis and kidney invasion.⁸ These gene regions are especially important in development of inflammation in urinary tract infections.³³ Also in our study, cnf-1 gene region was highly found in urine and hly gene region was only found in urine.

Although no statistically important relation found between carbapenem resistance and virulence factors in this study, the effect of beta-lactam resistance in virulence is known. The relation between the virulence factors and ESBL in E.coli were reported by many researchers.^34,35 Also, Arısoy et al. reported that increase of virulence genes were related with resistance to some antibiotics or sensitivity to others.¹³ In recent studies, a few mechanisms were focused for the relation and one of the mechanism is the plasmids carrying antibiotic resistance [³⁶] and others are porin loss³⁷, modifications in penicillin binding proteins and efflux pumps mechanism.³⁸ Efflux pumps are responsible for discharging of molecules containing virulence factors regulated by quorum sensing which has a positive effect on antibiotics resistance and virulence.^39,40E.coli strain 28 which previously reported with porin loss and OXA-48 resistance²⁵, was determined to have gene regions related with siderophore (aer, iutA) in this study. Similarly, virulence factors were highly found in carbapenem susceptible isolates, shows that other mechanisms may have an effect on the relation between carbapenem resistance and virulence. Therefore, determining of beta-lactam group resistance, these mechanisms should be taken into consideration.

CONCLUSIONS

Consequently, results demonstrated that virulence factors, antibiotic resistance, porin loss, multi drug efflux pump and quorum sensing molecules should be considered collective manner in the further studies about bacterial pathogenesis for developing effective treatments.

ACKNOWLEDGEMENTS

The data of this study have been excerpted from the PhD thesis of the author. The authors thank Assoc Prof Abbas Yousefi Rad, Assoc Prof Ayşe Esra Karakoç and Sefer Erman Yılmaz, MD for collecting clinical specimens, Onur Candan, PhD, for comments and suggestions and Çağla Kılıç MSc, for her contribution to this study. This work was financed by Hacettepe University Scientific Research Project grant 014D01601002 and The Scientific and Technolog ical Research Council of Turkey (TUBITAK) with PhD Scholarship (2211/C).

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Publication Dates

• Publication in this collection
  2017

History

• Received
  03 Feb 2016
• Accepted
  14 July 2016