A Surveillance of Antimicrobial Resistance Pattern and Virulence Factor in Uropathogenicescherichia Coli
1Debasish Sahoo, 2Ashoka Mohapatra, 1Rajashree Panigrahi, 1Purabi Baral*, 3Jatindra Nath Mohanty,1GoutamPattnaik, 4Mamata Bhat
1Department of Microbiology, IMS and Sum hospital, SOA deemed to be University, Bhubaneswar-751003, Odisha, India
2AIIMS Bhubaneswar Sijua, Patrapada, Bhubaneswar-751019
3Medical Research Laboratory, IMS and Sum hospital, SOA deemed to be University, Bhubaneswar-751003, Odisha, India
4Hi-Tech Hospital Road, Pandra, Rasulgarh, Bhubaneswar, Odisha 751025 Corresponding Author
Dr. Purabi Baral, 1Department of Microbiology, IMS and Sum hospital, SOA deemed to be University, Bhubaneswar-751003, Odisha, India
Mail [email protected]
Abstract
Escherichia coli (E.coli) is known to be the pathogen commonly isolated from those infected with urinary tract infections (UTIs). The aim of this study was to investigate the presence of E.coli virulence factor and antibiotics resistance pattern among clinical isolates from theurinary tract infections patient. 94 E.coliisolates from 166 UTI patients were taken as cases &
20 E.coliisolated from healthy individuals were taken as controls. Out of 94 isolates, 75(79.78%) were identified as UPEC in cases and 7(35%) were from 20 isolates in controls. Hemolysin production & cell surface hydrophobicity was found to be the major combination(36%) followed by serum resistance&hemolysinproduction (26.66%),serum resistance & cell surface hydrophobicity(22.66%). Most resistant drugs were Cotrimoxazole (65.95 %) followed byciprofloxacin (42.55%) &gatifloxacin(32.04%). Imipenem(92.56%) &amikacin(90.43%) were found to be highly sensitive drugs. Though CSH was major virulence factor among all, hemolysin production was detected predominantly in MDR strains(76.92%) as well as ESBL producers(51.28%). Interestingly also we observed that virulence factors production was inversely related to ESBL production. As most urovirulent strains express multiple virulence factors simultaneously, further studies on interaction of different virulence factors at molecular labelare necessary.
Keywords: Escherichia coli, MDR, virulence factor, uropathogenic, UTI
Introduction
Urinary tract infection (UTI) is an inflammatory response of theuroepithelium to bacterial invasion and is defined as the presence and multiplication of micro-organisms anywhere in the urinary drainage system, which is usually associated with bacteriuria and pyuria (1). It is characterized as upper UTI or lower UTI. Basing primarily on the anatomical location of the
infection, the lower urinary tract encompasses the bladder & urethra, and the upper urinary tract encompasses the ureter & kidneys (2).The upper urinary tract infections are most commonly ascending, i.e. they originate in the urinary bladder and ascend through the ureter to the kidney.
Normally the vesico-urethral valve prevents reflux of urine from the urinary bladder into ureters.
Individuals with urogenital anomalies or with overdistention of urinary bladder from out-flow obstruction, neurogenic malfunction or pressure from an enlarged uterus during pregnancy isparticularly susceptible to ascending UTI. Infection of renal pelvis (Pyelitis) &kidney (Pyelonephritis) are the most common complications (3).
UTIs can also be categorized into uncomplicated UTIs which mostly infect healthy individuals or those with no history of structural or neurological urinary tract abnormalities (3,4) and complicated UTIs which involve factors attenuating urinary tract or the host’s defense system including situations like urinary tract obstruction, urinary retention due to neurological condition, suppressed immune system, renal failure, kidney transplant, gestation and the existence of foreign bodies such as calculi, drainage devices or indwelling catheters (5,6).
Depends on the site of infection, UTIs are comprised of cystitis (bladder infection), pyelonephritis (kidney infection), prostatitis (prostate infection) and urosepsis, although bacteriuria is considered a probable typical symptom in all UTIs (7).
Uropathogenic Escherichia coli (UPEC) is considered as the dominant microorganism causing UTI. The microorganisms responsible for causing UTI are either Gram-negative or Gram- positive bacteria including Escherichia coli, Proteus mirabilis, Klebsiella species (Klebsiellapneumoniae), Serratiamarseciens, Citrobacter species, Gardnerellavaginalis, Pseudomonas aeruginosa, Enterobacter species (Enterococcus faecalis), Streptococcus group B (GBS), Staphylococcus species (Staphylococcus saprophyticus), Staphylococcus aureus, Mycoplasma species, Urea plasma species and Candida spp. (8-11).
Depending on which virulence factors are expressed, UPEC’s ability of causing symptomatic UTIs will be affected. The major virulence factors of E.coli are either associated with the surface of the bacterial cell or secreted and exported to their site of action. Type 1 fimbriae (fimH), pyelonephritis– associated pilus (pap), and S fimbrial adhesion (sfa) are the adhesion factors which boost colonization through various processes.
Since type 1 fimbriae is expressed both in pathogenic and commensal isolates, it is hard to resolve its role in human diseases. In fact, the frequency of fimH gene would not alter if the isolates causing UTI have a high or low virulence. Although it has been proven that P fimbriae are responsible for the increase in early colonization of tubular epithelium by some mechanisms concerning inter bacterial binding and biofilm formation, type 1 fimbriae mediate the colonization of the center of tubule. P fimbriae, encoded by pap (pyelonephritis–associated pilus) operon, are the most significant mannose–resistant adhesions expressed by E.Coli and will result in extra-intestinal infections (12)Keeping in view the above facts ,the present study is to isolate and identify Escherichia coli strains from clinically diagnosed cases of Urinary tract infection and to determine the virulence factors / markers like cell surface hydrophobicity, haemolysin
production, serum resistance &gelatinase production. Also we studied the drug resistance pattern & ESBL production.
Materials and Methods
The study groups comprised of hospitalized patients of all age groups attending our Medical college & Hospital with clinical signs of UTI like urgency, frequency, dysuria, pain abdomen, fever or asymptomatic cases with pyuria. 94 E.coliisolates from 166 UTI patients were taken as cases & 20 E.coliisolated from healthy individuals were taken as controls.
Inclusion criteria
Specimens were collected from clinically suspected cases of UTI irrespective of age and sex.
E.colithus isolated from the specimens were included for the study.
Exclusion criteria
Organisms other than Escherichia coli isolated from the specimens were excluded from the study.
Specimen was collected from urine, and stool sample of enrolled patient for this study and different growth media for microbial growth were prepared like CLED agar, Mac Conkey agar.
Then the standard protocol was used to identify the microbes followed by virulence markers analysis like the cell surface hydrophobicity, salt aggregation test, hemolysin test, Serum resistance, gelatinase production etc.
Drug sensitivity test
Mueller-Hinton agar plates were used for drug sensitivity test by modified Kirby Bauer disc diffusion method using the commercially available discs (HiMedia,Mumbai) like amikacin (30µg), cefpodoxime (30µg), ciprofloxacin(5µg), cotrimoxazole(25µg), gatifloxacin(5µg) gentamicin(30µg), imipenem(30µg), nitrofurantoin (300µg) &norfloxacin (10µg). After overnight incubation, the zone size was interpreted as sensitive, intermediate or resistant according to NCCLS criteria.(Collee JG et al,1996).
Strains which were resistant to ≥ 2 of the above antibiotics were considered as multidrug resistant(MDR) strains.
Tests for extended spectrum β-lactamase production
A. Screening methods (standard disk diffusion) - Screening for ESBL production was done by using third generation cephalosporins (3GC) viz., ceftazidime, cefotaxime&
ceftriaxone each 30mcg/disc as per NCCLS recommendations. Zone diameters were read using NCCLS criteria. Resistance or decreased susceptibility to any of the 3GC antibiotics i.e an inhibition zone of ≤ 22 mm for ceftazidime and ≤ 27 mm for cefotaxime was considered as potential ESBL producer and was confirmed by phenotypic confirmatory testing i.e Double Disc Diffusion Synergy Test(DDST) ( Jain A et al,2003).
B. Confirmatory methods (Double Disc Diffusion Synergy Test /DDST) - Ceftazidime (30μg) vs. ceftazidime/clavulanic acid (30/10 μg) and cefotaxime (30 μg) vs.
cefotaxime/clavulanic acid (30/10 μg) were placed onto Mueller Hinton agar plate at
a distance of 30mm apart, lawned with the test organisms and incubated at 37°C overnight. Regardless of zone diameters, ≥ 5 mm increase in the zone diameter of an antimicrobial agent tested in combination with clavulanic acid vs. its zone size when tested alone, was confirmed as ESBL producers (Sharma et al,2007).
Stastistical analysis
The chi square test was used to compare the occurrence of virulence markers in both cases and controls. P value less than 0.05 was considered significant.
Results and observations
166 urine samples from UTI cases and 20 stool samples from healthy controls were screened for uropathogenicE.coli. 56.62 % isolates of E.coli were obtained from urine & 100 % from stool samples. Majority of the isolates were obtained from female(60.64 %) as compared to males(39.36 %) Among cases 79.78 % strains were found to be UPEC where as 35 % were identified from controls which was found to be highly significant(p<0.005).Cell surface hydrophobicity (CSH) was found to be major virulence marker (80% ) followed by hemolysin production (52%) and others (Table 1). Seven isolates were found to be UPEC Out of 20 controls, among which CSH(20%) was most predominant marker followed by HP(10%) &
others (Table 2).
Table 1: Distribution of different virulence markers among UPEC(n=75)
Virulence markers No of isolates %age Cell surface hydrophobicity (CSH) 60 80
Haemolysin production(HP) 39 52
Serum resistance(SR) 27 36
Gelaninase production(GP) 7 9.33
Table 2: Distribution of Virulence markers among controls( n=20)
Virulence markers No of isolates %age Cell surface hydrophobicity (CSH) 4 20
Haemolysin production(HP) 2 10
Serum resistance(SR) 1 5
Gelaninase production(GP) 0 0
All the virulence markers were predominantly detected in females. Hemolysin production + cell surface hydrophobicity combination was observed most predominantly ( 36 %) among the isolates followed by serum resistance + hemolysin production (26.66%) and others.
Gelatinase production was observed alone and was not associated with any other virulence markers (Table 3).
Table 3: Distribution of UPEC in different combinations(n=75) Combination of virulence
markers n %
CSH + HP 27 36
SR + HP 20 26.66
SR + CSH 17 22.66
SR + GP 0 0
CSH + GP 0 0
HP + GP 0 0
SR + CSH + HP 16 21.33
SR + CSH + HP + GP 0 0
CSH-Cell surface hydrophobicity,HP- Hemolysinproduction,SR- Serum resistance,GP- Gelatinase production.
Cotrimoxazole was found to be most resistant (65.95 %) among the routinely used drugs, followed by ciprofloxacin(42.55%), Gatifloxacin(34.04%) and others. Imipenem(92.56%) was the most sensitive drug followed by amikacin (90.57 %).(Table 4).
Table 4: Distribution of drug resistance pattern (n=94)
Drugs Ak Cep Gen Cip Gat Nf Nx Co I
Total 9 25 24 40 32 10 20 62 7
% age 9.57 26.59 25.53 42.55 34.04 10.63 21.27 65.95 7.44
Hemolysin production was the predominant virulence marker (76.92%) among the MDR strains followed by cell surface hydrophobicity (48.33%) and others (Table 5)
Table 5: Correlation of MDR strains with UPEC
Virulence Markers MDR Strains % age
Cell Surface Hydrophobicity(n=60) 29 48.33
Hemolysin Production(n=39) 30 76.92
Serum Resistance(n=27) 10 37.03
Gelatinase Production(n=7) 2 28.57
Out of the 75 UPEC, 48( 64%) were screened to be ESBL producers, among which 91.66%
were confirmed by Double Disc Diffusion Synergy Test (DDST). (Table 6)
Table 6: Correlation of ESBLproducers with UPEC
Virulence Markers ESBL Producers % age
Cell Surface Hydrophobicity(n=60) 18 30
Hemolysin Production(n=39) 20 51.28
Serum Resistance(n=27) 6 22.22
Gelatinase Production(n=7) - 0
Table 7: Association of ESBL producers & multiple virulence factors
Hemolysin production was also the predominant virulence marker (51.28%) among ESBL producers followed by cell surface hydrophobicity(30%) and others (Table 6). As the number of virulence factors increases, rate of ESBL production decreases (Table 7).
Discussion
UTI is one of the commonest problem encountered by the primary as well as tertiary care hospitals. Considering the high degree of morbidity and mortality of UTIs, the subject of UPECwas receiving increasing attention.Cell morphology and molecular biology studies have revealed that UPEC express several surface structures and secrete protein molecules. Some of them are cytotoxic and are peculiar to the strains of E. coli causing UTI. Hence, it is important to identify UPEC from non-UPEC isolates in the urinary samples. Ninetyfour (56.62%) E.colistrainswere isolated from 166 urine samples & 20 strains were isolated from stool samples of 20 healthy individuals. In the present study, out of 94 isolates studied, 57(60.64%) were from females and 37(39.36%) from males with a male to female ratio of 0.65:1, which correlates well with the ratio of 0.72:1 by Karabiber et al during their study in 1992 (13).However wide variation of M: F ratio were reported in various studies like Ruiz et al, 2002 &MatijaRijavec et al,2008 (14,15). This femaleprepodencemay be partially due to short &
wider female urethra & its proximity to anus which allow bacteria to travel up & cause infections (1).
Multiple virulence factors ESBL+ ESBL-
Present 16(36.36%) 32(64%)
Absent 28(63.63%) 18(36%)
Total 44 50
In our study, 75(79.78%) of isolates were identified to be UPEC from 94 strains. Ruiz et al, 2002 (14) had also reported 81% & 82 % of UPEC in their studies respectively.Wide variation in identification UPEC were observed in different studies like Heather et al,1980 (47%); Raksha et al, 2003 (68.63%)&Vagarali et a1,2008 (25%) (16,17,18) When virulence of the isolates from controls were studied, only 35% were identified to be UPEC in contrast to the cases(79.78%).This difference when analysed statistically was found to be highly significant (p<0.001).The study conducted by Karabiber et al in 1992 (13) was also identified 31% UPEC from control strains.
Though hemolysin production is not essential for the establishment of acute pyelonephritis, it might contribute to tissue injury, survival in the renal parenchyma and entry into blood stream.It is also an important virulence factor in recurrent UTI.So it can guide the clinician in deciding whether to treat for E.colicolonization or not by indicating which isolates pose a higher risk of causing a symptomatic UTI under certain conditions. In our study 39(52%) of UPEC were showing hemolysin production(Table-I). This is nearer to 43% & 41.36% in the studies of Raksha et al, 2003 (17). In the study of Ruiz et al ,2002 (14), 81 % UPEC were producing hemolysin.There was a wide variation inhemolysin producing property among UPEC were reported in studies of Heather et al, 1981 (28%), Karabiber et al,1992 (21%), (13,16). This was probably due to the possession of a gene sequence named hlyA which encodes the α- hemolytic activity of the E.colistrains isolated from urinary tract infections(Joaquim Ruiz et al, 2002) (14).
The high hydrophobicity of the bacterial cell surface promotes their adherence to various surfaces like mucosal epithelial cells by the crystalline surface layer 's' present on both Gram negative &Gram positive organisms. Hydrophobicity is a recently described novel virulence mechanism by Esch.coli. It was observed in 80% of UPEC in our study(Table-I) .In 1990, Brauner et al were also identified 82% of UPEC showing hydrophobic property. Serum resistance is the property by which the bacteria resist killing by normal human serum due to lytic action of complement system. Isolates from patients with pyelonephritis, cystitis and bacteraemia are typically serum resistant whereas patients with asymptomatic bacteriuria have serum sensitive strains. There is a strong correlation between serum resistance and the ability of a variety of gram negative bacteria to invade & survive in blood stream. Among the isolates, 27(36%) were identified to have serum bactericidal activity in our study(Table-1) which correlates with the study of Raksha et al in 2003(32.72%) (17). Gelatinase is a protease enzyme produced by some of the virulent E.coli strains. In our study 9.33 % were identified to possess this enzyme(Table-1), which is almost nearer to study conducted by Sharma et al in 2007 (6.9%) (19).
Uropathogenicity is a multifactorial phenomenon. The virulence factors function additively or synergistically in overcoming the normal host defences. The combination of hemolysin production & cell surface hydrophobicity was observed in 36% isolates followed by serum resistance &hemolysin production( 26.66%) and serum resistance & cell surface hydrophobicity combination(22.66%).(Table-3) This correlate nearly with the study of Raksha et al in
2003(17). The strains with a more extensive virulence factors are more effective pathogens and the compromising host conditions decrease the need for multiple virulence factors in strains causing serious infections.Themajority of the uropathogenic strains possessed one or more of the virulence properties examined, while such strains were in the minority in the faecal group.
Presuming all faecal strains have the same opportunity to enter the bladder, those with more than one virulence property are more likely to cause infection.
Among the routinely used drugs, most resistant drug was Cotrimoxazole (65.95 %) followed by ciprofloxacin &gatifloxacin. Imipenem(92.56%) &amikacin(90.43%) were found to be highly sensitive drugs.(Table-4) The greater prevalence of resistance to common antibiotics has also been reported by other workers. This may be due to the dissemination of antibiotic resistance among hospital isolates of E. coli . These results are consistent with the previous studies bySuman E et al in 2001 (20). We observed a high rate of ESBL production by the isolates which may be due to the selective pressure imposed by extensive use of antimicrobial agents.
Out of 75 UPEC isolated, 48(64%) were screened as ESBL producers by standard disc diffusion method & among them 44(91.66 %) were confirmed by Double disc diffusion synergy test( DDST)(Table-6). Sharma et al in the year 2007 (19) had alsocarriedout similar study on ESBL production where 51.4% were screened as ESBL producers, among which 92.9% were confirmed. The indiscriminate use of cephalosporins may be responsible for the high rate of ESBL production. For predicting ESBL production, it is important to mention that for screening test, negative results are a better guide than positive results. Therefore, confirmation of all positive results by screening should be done to prevent unnecessary
avoidance of conventional β-lactams use.
Majority of ESBL negative strains(64%) were produce multiple virulence factors whereas most of the ESBL producers (63.63%) did not produce (Table-5).This correlates with the study of Sharma et al,2007 (19).These result support the hypothesis that although virulence factors and antibiotic resistance may confer increased fitness for infections , they may do so via mutually exclusive pathways and in distinct populations. Hemolysin production was most predominant among MDR strains and ESBL producers((76.92%, 51.28%
respectively) followed by CSH(48.33%, 30% respectively) (Table-7) which fits with the study by Sharma et al,2007 (19).
Conclusion
As Escherichia coli has the ability to adapt and survive in various conditions by delivering numerous harmfulness factors which help in developing drug resistance, Periodic review and formulation of antiobiotics policy are needed for control of acquisition of drug resistance.
Moreover the pathogenic potentiality of E.coli in UTI differs in population depending on the possession of various virulence factors and susceptibility to antimicrobials. Hence this has to be taken into account while developing guidelines for management of UTI. As most urovirulent strains express multiple virulence factors simultaneously, further studies on interaction of different virulence factors at molecular level are necessary. Understanding the antibiotic
resistance in various geological regions is significant; consequently, the best antibiotic is chosen to treat urinary tract infections. Meanwhile, it is critical that the antibiotic of choice would not increase the antibiotic resistance. To advance improvement in future investigations, it is prescribed to gather to collect more E. coli isolates from different hospitals and to investigate more virulence factors among these isolates. Furthermore, it is all around informed to contemplate the expression of the virulence genes using real time PCR.
Conflict of Interest: No Funding agencies: NA
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Legends
1. Table 1: Distribution of different virulence markers among UPEC(n=75) 2. Table 2: Distribution of Virulence markers among controls ( n=20) 3. Table 3: Distribution of UPEC in different combinations(n=75) 4. Table 4: Distribution of drug resistance pattern (n=94)
5. Table 5: Correlation of MDR strains with UPEC 6. Table 6: Correlation of ESBL producers with UPEC
7. Table 7: Association of ESBL producers & multiple virulence factors
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