MOLECULAR DETECTION AND ANTIMICROBIAL ACTIVITY OF ENTEROCOCCUS FAECALIS AND ENTEROCOCCUS FAECIUM ISOLATED FROM URINARY TRACT INFECTION (UTI) PATIENTS, ANIMALS AND POULTRY

                                                                                                                 AssiutUniversity web-site: www.aun.edu.eg

MOLECULAR DETECTION AND ANTIMICROBIAL ACTIVITY OF ENTEROCOCCUS FAECALIS AND ENTEROCOCCUS FAECIUMISOLATED FROM URINARY TRACT INFECTION (UTI) PATIENTS, ANIMALS AND POULTRY

ALSHIMAA A. HASSANIEN

Department of Zoonoses, Faculty of Veterinary Medicine, Sohag University, Egypt.

Received: 8 March 2016;      Accepted: 12April2016

INTRODUCTION

Recent years have witnessed increased interest in enterococci not only because of their ability to cause serious infection like endocarditis, bacteremia, intra-abdominal and urinary tract infection (UTI), but also because of their increasing resistance to many antimicrobial agents (Desai et al., 2001). In humans, as well as in other mammals and birds, enterococci are mainly found in the gastrointestinal tract as commensals but may become opportunistic pathogens in individuals with serious diseases whose immune systems are compromised and in patients who have been hospitalized for prolonged periods or who have received broad-spectrum antimicrobial therapy (Gonzalo et al., 2013). Antibiotics may promote colonization and infection with multidrug resistant enterococci by at least two mechanisms; First, many broad spectrum antibiotics have little or no anti-enterococcal activity, and administration commonly leads to overgrowth of susceptible or resistant enterococci. Second, most antibiotics substantially

Corresponding author: Dr. ALSHIMAA A. HASSANIEN

E-mail address: hassanien2008@yahoo.com

Present address: Department of Zoonoses, Faculty of Veterinary Medicine, Sohag University, Egypt.

reduce the normal resistance of the intestinal tract to colonization by exogenous organisms (Miller et al., 2014). Therefore, the selective pressure caused by the intensive use of antimicrobial agents in human and veterinary medicine, contributed to the emergence and wide spread of resistance mechanisms in bacteria of different ecosystems (Lebreton et al., 2013). Furthermore, anti- microbial-resistant enterococci in animals are likely to serve as a reservoir from which resistance genes are transferred to enterococci in humans, either through human consumption of food of animal origin, by direct contact between animals and humans, or via the environment. (Heuer et al., 2006).

Enterococcal surface protein encoded by the chromosomal esp associated with increased virulence, colonization and persistence in the urinary tract (Shankar et al., 2001), and biofilm formation which could lead to resistance to environmental stresses, and adhesion to eukaryotic cells of the urinary tract (Borgmann et al., 2004). Therefore, disruption of the esp gene impairs the ability of E. faecalis to form biofilms (Latasa et al., 2006). In addition, E. faecium strains that carry the esp gene have higher conjugation rates than strains that do not possess this gene. The aim of this study was to detect the extent of E. faecalis and E. faecium in UTI patients and their reared animals and poultry and detect some virulence factors of enterococci as antimicrobial resistance and esp gene presence.

MATERIALS AND METHODS

1-    Sampling

• Human samples

Between September 2014 and December 2015 a total of 95 urine samples were collected from patients suffering from urinary tract infections admitted to outpatient clinics of private and governmental hospitals in Sohag city, Egypt. Urine samples were immediately transported to the laboratory in the faculty of veterinary medicine, SohagUniversity for microbiological isolation and identification of Enterococcus faecalis and Enterococcus facium. All patients were asked about whether they reared animals and / or poultry in their houses, infection recurrence and antimicrobial used.

• Animals and poultry samples

52 rectal and 50 cloacal swabs were collected from different animals (18 sheep, 21 goats and 13 cattle) and /or poultry (35 chicken and 15 duck) reared in 11 patient’s households whose urine samples give positive results for Enterococcus faecalis and Enterococcus facium. The number of recteal or cloacal swabs collected from animals or poultry from each household is ranged from 3 to 5 samples for each animal and / or poultry species.

2-    Isolation and identification of Enterococci

The samples were inoculated into enterococcus selective broth and incubated at 37ºC for 24 hrs, a loopful from incubated tubes was streaked onto KF Streptococcal agar (TM media, India) and incubated at 37ºC for 48 hrs, red colonies presumptive to be Enterococci were transferred to nutrient agar slants for further identification of Enterococcus species according to Morrison et al. (1997) and Manero and Blanch, (1999).

3-    Molecular detection

• Genomic DNA extraction

DNA was extracted from all isolates of enterococci using the QIAamp DNA mini kits (QIAGEN, Germany, No. 69504) in accordance with the manufacturer’s instructions.

• Detection of  E. faecalis and  E. facium

Multiplex PCR for detection of D-alanine-D-alanine ligase (ddl) of E. faecalis and E. faecium was done as described by Dutka-Malen et al. 1995 with modifications as the following; initial denaturation step at 94°C for 5 min; 30 cycles of amplification (denaturation 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min); and a final extension at 72°C for 5 min. The PCR products were electrophoresed through a 1% agarose gel stained with ethidium bromide and transilluminated under UV light. 100 pb DNA ladder (Norgen biotek, Canada) was used as a marker.

• Detection of esp gene

PCR was carried out to detect esp gene among all isolates of Enterococcus faecalis and Enterococcus facium of UTI patients and their reared animals and poultry as reported by Vankerckhoven et al. (2004) but conditions have been optimized for esp gene, as initial activation step at 95°C for 15 min, followed by 30 cycles at 94°C for 1 min, annealing at 56 °C for 1 min. and extension at72 °C for 1 min followed by final extension cycle at 72 °C for 10 min.

Sequence of primers used for detection of E.faecalis, E.faecium and esp gene.

4-    Antimicrobial sensitivity test

The disk diffusion method of antimicrobial sensitivity test was performed according to Clinical and Laboratory Standards Institute (CLSI) guideline (2009) using antimicrobial discs of (Oxid, UK). The all isolated strains of E. faecalis and E. facium recovered from UTI patients and their reared animals were tested against Amikacin (AK) 30µg, Amoxycillin / clavulanic acid (AMC) 30 µg, Ciprofloxacin (CIP) 5 µg, Vancomycin (VA) 30 µg, Spiramycin (SP) 100 µg, Gentamicin (CN) 120, Ceftriaxone (CRO) 5µg, Nitrofurantoin (F) 300 µg, Tetracycline (TE) 30 µg and Neomycin (N) 30µg.

RESULTS

Table1: PCR detection of E. faecalis and E. faecium among UTI patients.

Table 2: PCR detection of E.faecalis and E.faecium among animals and poultry in patient’s households.

Table 3: Antimicrobial profile of E.faecalis and E.faecium isolated from UTI patients.

Amikacin (AK), Amoxycillin/ clavulanic acid (AMC), Ciprofloxacin (CIP, Vancomycin (VA), Spiramycin (SP), Gentamicin (CN), Ceftriaxone (CRO),  Nitrofurantoin  (F), Tetracycline (TE ), Neomycin (N)

Table4: Antimicrobial profile of E.faecalis and E.faecium isolated from animals and poultry.

Table 5: Frequency distribution of esp gene among E.faecalis and E.faecium isolated from human, animals and poultry.

Table 6: Frequency distribution of esp gene among antimicrobial resistant strains of E.faecalis and E.faecium isolated from human, animals and poultry.

                                 Figure (1)                                                                   Figure (2)       

Figure (1): Agarose gel electrophoresis of multiplex PCR amplification products using specific ddl E. faecalis primer of E.faecalis and ddl E. faecium primer of E.faecium. Lane M: 100 bp ladder as molecular DNA marker, lane1and lane 2: positive E. faecium, lane 3, lane 5, lane 6 and lane 7: positive E.faecalis, lane 4: Negative

Figure (2): Agarose gel electrophoresis of PCR amplification products using specific esp gene of E.faecalis and E. faecium. Lane M: 100 bp ladder as molecular DNA marker, lane 2, lane 3, lane 4, lane 5, lane 6, lane 7, lane 8 and lane 10:  positive esp gene, lane 1, lane 9 and lane 11: Negative esp gene.

DISCUSSION

Microbiological and PCR analysis of 95 urine samples collected from patients suffering from UTI recurrence revealed detection of E.faecalis and E.faecium in 13 (13.7%) patients; E.faecalis wasslightly higher than E.faecium  with percentage of 8.4% and 5.3% respectively (Table1). In comparison with Gonzalo et al. (2013) and Sharifi et al. (2013) who recorded higher percentage of infection; enterococci in the present study not reflect the true incidence of infection but it definitely suggest the increased frequency of their isolation from UTI patients and this variation may be due to the difference in the study population.

The majority of patients infected with enterococci in the present study reared animals and / or poultry in their households (Table1) which enhance the probability of zoonotic transmission of enterococci from animals to human. From the results in Table 2 it is clear that E. faecalis and E. faecium were isolated by microbiological and PCR methods from 15 (14.7%) out of 102 fecal and cloacal samples of reared animals and poultry in 11 patient's households with highest percentage in cattle (30.8%), followed by sheep (16.7%), goat (14.3%), ducks (13.3%) and chicken (11.4%), therefore close contact with animals may play a role in enterococcal infection but exposure to infection outside the household environment cannot be excluded, so further epidemiological studies are needed to investigate the risk factors of enterococcal infection.

Arias et al. (2010) illustrated that enterococci possess intrinsic or acquired resistance to several antimicrobials, such as glycopeptides, b-lactams, and fluoroquinolones, and can exhibit high levels of resistance to aminoglycosides, leading to drastically reduced therapeutic options for patients infected with enterococci, owing to the lack of antimicrobial policy and the massive use of antibiotics both in the human health care system and agriculture. Furthermore, several antimicrobial agents that are used in animals belong to the same class of antimicrobial agents of clinically important for human therapy, thus antimicrobial resistant enterococci may frequently be transferred from animals to humans either by ingestion of contaminated food or from the environment. (Heuer et al., 2006).

Results in Table 3 revealed that E. faecalis and E. faecium isolates recovered from UTI patients exhibited higher resistance to the most common antimicrobials such as amikacin, gentamicin and tetracycline with percentage of (69.2%) followed by Ceftriaxone (38.5%), spiramicin and nitrofurantoin (30.8%), vancomycin and ciprofloxacin (23.1%), amoxycillin/ clavulanic acid (15.4%), while lower resistance to neomycin (7.7%) associated with lower use of this antibiotic in human therapy. Furthermore, 10 (76.9%) out of 13 E. faecalis and E. faecium strains isolated from clinical human samples were multidrug resistant to at least three or more unrelated antimicrobials led to recurrence of infection among the infected patients, this results goes parallel to Sharifi et al. (2013). Table 4 showed that E. faecalis and E. faecium strains isolated form fecal and cloacal samples of reared animals and poultry in 11 patient's households were frequently resistance to the similar antimicrobials which used for human medicine with highest proportion for gentamicin and tetracycline (93.3%) followed by vancomycin (53.3%), amikacin and spiramicin (40%), Ceftriaxone and neomycin (26.7%), ciprofloxacin (20%), nitrofurantoin (13.3%) and amoxycillin/ clavulanic acid (6.7%). In addition, multidrug resistant to at least three or more unrelated antimicrobials were detected in 13 (86.7%) out of 15 strains of E. faecalis and E. faecium recovered from reared animals and poultry in 11 patient's households. Therefore, concerns about public health issues evoked by exchanging antimicrobial-resistant and virulent enterococci between animals and human beings have increased (Ghosh et al., 2011).

Harada et al. (2005) elucidate that although esp gene in enterococci was initially found only in hospital derived human isolates of enterocicci, the esp gene was later observed in human and animal isolates in community settings. Results in Table 5 showed that 11 (84.6%) out of 13 strains of E. faecalis and E. faecium  isolated from UTI patients possess esp gene, this result is lower than that recorded by Vankerckhoven et al., 2004 and Sharifi et al., 2013 and higher than that reported by Strateva et al., 2016. In contrast, Shanker et al., 1999 who revealed failure of detection of esp genein E. faecium. In addition, 9 (60%) out of 15 strains of E. faecalis and E. faecium  isolated from reared animals and poultry in patient´s household were harbor esp gene, this result is higher than the results obtained by Klibi et al., 2014. In contrast, Kown et al., 2012 and Sˇeputiene et al., 2012 who detect esp gene in E. faecalis only. The presence of multidrug resistance among our study may be related to the higher incidence of esp gene in the resistant isolates of E.faecalis and E.faecium in both UTI patients and their reared animals and poultry (Table 6), since the presence of this gene promote adhesion, colonization and evasion of the immune system, and to play some role in antibiotic resistance (Moreno et al., 2006).

CONCLUSION

The emergence of antimicrobial resistance enterococci among UTI patients and their reared animals and poultry emphasizes the need to investigate their ecology, epidemiology and virulence.

REFERENCES

Arias, C.; Contreras, G. and Murray, B. (2010): Management of multidrug-resistant entero-coccal infections. Clin Microbiol Infect., 16: 555–562.

Borgmann, S.; Niklas, D.; Klare, I.; Zabel, L.; Buchenau, P.; Autenrieth, I. and Heeg, P. (2004): Two episodes of vancomycin resistant E. faecium outbreaks caused by two genetically different clones in a newborn intensive care unit. Int. J. Hyg. Environ Health., 207: 386–389.

Clinical and laboratory standards institute (CLSI) (2009): Performance Standards for antimicrobial susceptibility testing: Nineteenth Informational Supplement M100-S19. CLSI, Wayne, PA, USA.

Desai, D.; Pandit, M.; Mathur, A. and Gogate, A. (2001): Prevalence, identification and distribution of various species of enterococci isolated from clinical specimens with reference to urinary tract infection in catheterized patients. Indian J. of Med. Microbiol.; 19(3): 132-137.

Dutka-Malen, S.; Evers, S. and Courvalin, P. (1995): Detection of glycopeptides resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol., 33(1): 24-27.

Gonzalo, C.; Marisa, M.; Sergio, P.; Rosa I. A.; Rau´ l. A.; Johannes, H.; Yolanda, L. (2013): Comparison of E. faecium and E. faecalis strains isolated from water and clinical samples: Antimicrobial susceptibility and genetic relationships. PLOS ONE | www.plosone.org., 8,4: e59491.

Ghosh, A.; Dowd, S. and Zurek, L. (2011): Dogs leaving the ICU carry a very large multi-drug resistant enterococcal population with capacity for biofilm formation and horizontal gene transfer. PLOS ONE 6,7: e22451.

Harada, T.; Tsuji, N. and Otsuki, K. (2005): Detection of the esp gene in high level gentamicin resistant E.faecalis strains from pet animals in Japan Vet. Microbiol., 106: 139–143.

Heuer, O.; Anette, M.; Peter, C. and Henrik, C. (2006): Human health hazard from antimicrobial resistant enterococci in animals and food. Food safety, 43: 911-916.

Klibi, N.; Rim. A. and Francesca, B. (2014): Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia. Ann Microbiol., 65:     695-702

Kwon, K.; Hwang, S.; Moon, B.; Young, P.; Sook, S.; Cheol-Yong, H. and Yong, H. (2012): Occurrence of antimicrobial resistance and virulence genes, and distribution of enterococcal clonal complex 17 from animals and human beings in Korea. J. of Veterinary Diagnostic Investigation, 24(5) 924–931.

Latasa, C.; Solano, C.; Penade´, S.J. and Lasa, I. (2006): Biofilm associated proteins. Comptes Rendus Biologies., 329: 849- 857.

Lebreton, F.; Van Schaik, W. and McGuire, A. et al. (2013): Emergence of epidemic multidrug-resistant E. faecium from animal and commensal strains. MBIOǀ www. mbio.asm. org., 4, 4: e00534.

Manero, A. and Blanch, A. (1999): Identification of Enterococcus spp. with a biochemical key. Applied and environmental microbiology, 65: 4425–4430.              

Miller, W.; Munita, J. and Arias, C. (2014): Mechanisms of antibiotic resistance in enterococci. Expert Rev. Anti Infect. Ther., 12(10): 1221–1236. 

Moreno, M.; Sarantinopoulos, P.; Tsakalidou, E. and DeVuyst, L. (2006): The role and application of enterococci in food and health. Int. J. of Food Microbiol., 106: 1–24.

Morrison, D.; Woodford, N. and Cookson, B. (1997): Enterococci as emerging pathogens of humans. J. Appl. Microbiol., 83: 89-99.        

Sˇeputiene, V.; Bogdaite, A.; Ruzˇauskas, M. and Suzˇiede˙liene, E. (2012): Antibiotic resistance genes and virulence factors in E. faecium and E. faecalis from diseased farm animals: pigs, cattle and poultry. Polish J. Vet. Sci., 15: 431-438.             

Shankar, V.; Baghdayan, M.; Huycke, G.; Lindahl and Gilmore, M. (1999): Infection-derived E.faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect. Immun., 67: 193–200.

Shankar, N.; Lockatell, A.; Baghdayan, C.; Drachenberg, M.; Gilmore, M. and Johnson, D. (2001): Role of E. faecalis surface protein esp in the pathogenesis of ascending urinary tract infection. Infect. Immun., 69: 4366–4372.

Sharifi, Y.; Alka, H.; Reza, G.; Behrouz, N.; Mohammad, A.;  Mortaza, M. and Ahad, B. (2013): Virulence and antimicrobial resistance in enterococci isolated from urinary tract infections. Advanced Pharmaceutical Bulletin, 3(1), 197-201.

Strateva, T.; Atanasova, D.; Savov, E.; Petrova, G. and Mitov, I. (2016): Incidence of virulence determinants in clinical E. faecalis and E. faecium isolates collected in Bulgaria. Braz J. Infect dis., 548.

Vankerckhoven,V.; Tim, V.; Carl, V.; Christine, L.; Sabine, C.; Rosaria, R.; Daniela, J. and Herman, G. (2004): Development of a Multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J. Clin. Microbiol., 42: 4473–4479.