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Environmental Enterococci: I. Prevalence of virulence, antibiotic resistance and species distribution in poultry and its related environment in Karachi, Pakistan ARTICLE in LETTERS IN APPLIED MICROBIOLOGY · DECEMBER 2013 Impact Factor: 1.75 · DOI: 10.1111/lam.12208
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Letters In Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Environmental enterococci: I. Prevalence of virulence, antibiotic resistance and species distribution in poultry and its related environment in Karachi, Pakistan S.A. Ali*, K.A. Hasan*, H. Bin Asif and A. Abbasi International Center for Chemical and Biological Sciences (ICCBS), HEJ Research Institute of Chemistry, University of Karachi, Karachi, Pakistan
Significance and Impact of the Study: This study demonstrates that poultry environment of Karachi city harbours a diverse reservoir of Enterococcus spp. with multiple antibiotic resistance and virulence determinants. It is expected that the results will help in assessing the impact of multiple antibiotic resistance and virulent enterococci on public health, improvement of farm management practices and preventing their dissemination. Our findings strongly suggest the need for reducing antibiotic usage during poultry production, particularly those that are being used to treat human infections.
Keywords Box-PCR, enterococci, environmental indicator organism, multiple antibiotic resistance, multiple virulence determinants, multiplex PCR. Correspondence Syed A. Ali, International Center for Chemical and Biological Sciences (ICCBS), HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan. E-mails:
[email protected];
[email protected] *Shared first authors. 2014/1258: received 24 June 2013, revised 21 November 2013 and accepted 5 December 2013 doi:10.1111/lam.12208
Abstract Enterococci are well-established causative agents of a number of diseases in humans and animals alike. A total of 1047 isolates (838 presumptive enterococci and 209 non-enterococci), related to poultry environment (faeces/ manure, feed, water and air), were evaluated for species diversity, virulence potential, antibiotic resistance and associated threats. Multiplex PCR of 204 presumptive enterococci using genus (16s rDNA)- and species-specific (superoxide dismutase) primer set leads to identification of six Enterococcus spp., i.e. Ent. faecium, Ent. faecalis, Ent. mundtti, Ent. hirae, Ent. gallinarum and Ent. casseliflavus. BOX-PCR analysis of the representative strains confirmed inter-strain variation among species. Although enterococci generally appear to be catalase negative, Ent. faecalis from some faecal, water and air samples showed catalase production. In vitro assays identified poultry environment as a reservoir of MVD and MAR enterococci and non-enterococci. In addition to vancomycin, high-level-ampicillin-, clarithromycin-, erythromycin-, kanamycinand gentamicin-resistant enterococci and non-enterococci are also indicated. Our results show that virulence potential and resistance vary with the source of isolation. Our studies on MAR and MVD enterococci in environment, especially in air and water, clearly indicate the need for a serious commitment to deal with pathogenic enterococci associated with poultry for preventing transmission of infectious agent to humans.
Introduction Enterococcus spp. are Gram-positive, non-spore-forming facultative anaerobes having a high tolerance to harsh conditions, such as temperature (10–45°C), pH (96), hyperosmolarity (65% NaCl) and prolonged desiccation, but lack oxidase and catalase activity although with some exceptions (Frankenberg et al. 2002; Moraes et al. 2012). Also, resistance to physical and chemical agents such as Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
UV radiation, heavy metals, ethanol, azide, detergents, sodium hypochlorite and hydrogen per oxide is as en suite capability of this genus (Kakinuma 1998; Giard et al. 2001; De Niederh€ausern et al. 2013). Enterococci are mostly found in gastrointestinal tract (GIT) of humans and animals, but frequent reports have suggested their presence in extraenteral environment, as they have been isolated from sewage, soil, plants, poultry, dairy and meat products and even from the sea water (Foulquie Moreno 1
Environmental enterococci in poultry-related environment
et al. 2006). In addition to this, ability of the organism to survive in very hostile environment provides great temptation to be selected as a faecal contaminant indicator. Enterococcus spp. are the most frequent causative agents of a wide variety of human infections occurring in blood stream, heart, abdomen, urinary tract, etc., with Enterococcus faecalis being responsible for at least 70–80% of the cases (Poulsen et al. 2012). Also as a nosocomial pathogen, it causes ear and eye infections, meningitis, burns wounds and abscess, paranasal sinuses, etc. (Kapoor et al. 2005). Among Enterococcus spp., Ent. faecalis and Ent. faecium are the most important and have been isolated from both healthy and diseased individuals. Additionally, Ent. gallinarum, Ent. avium, Ent. hirae, Ent. casseliflavus, Ent. durans and Ent. cecorum have also been isolated from patients with a range of infections (Greub et al. 1997). Furthermore, isolation of Ent. cecorum from patients suggests their possible transfer route from poultry (De Baere et al. 2000). The acquisition of virulence, resistance to antibiotics and infection management caused by enterococci are serious issues for health occupational and scientists. Enterococcus spp., the key contributor for acquiring and disseminating antibiotic resistance, have been isolated from poultry meat, feed and other food-chain stuff (Kaszanyitzky et al. 2007). Poultry, which harbours a wide range of Enterococcus spp. such as Ent. avium, Ent. casseliflavus, Ent. cecorum, Ent. durans, Ent. gallinarum, Ent. hirae and Ent. malodoratus, is itself a target of many diseases; for instance, Ent. cecorum causes arthritis, spondylitis, femoral head necrosis, osteomyelitis, lameness and paralysis of broilers (Stalker et al. 2010; Velkers et al. 2011). Enterococcus faecium, Ent. durans and Ent. hirae are reported to cause endocarditis, bacteraemia, encephalomalacia and focal necrosis of the brain of young chicks, while Ent. faecalis causes ascites in hens and pulmonary hypertension in broilers (Velkers et al. 2011). Karachi, the largest and most thickly populated city of Pakistan, serves as a business and financial support centre. A research survey conducted by GALLUP (2008-9) indicates that 45% of population prefers chicken in their daily diet and therefore, monitoring of virulent and resistant enterococci in poultry is of utmost importance to ensure food safety and public health (Expert Panel on Antibiotic Resistance 2005). However, no surveillance data of Enterococcus spp. associated with poultry environment have been made in this city. Thus, our present study on Enterococcus spp. associated with livestock (poultry and its related environment) is expected to provide an insight into the species biodiversity, resistance, use of antibiotics in farm management practices and virulence potential acquired by enterococci. Our results clearly indicate that a serious commitment is required to deal with pathogenic 2
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enterococci associated with poultry for preventing transmission of infectious agent to humans. Results and discussion A total of 1047 isolates (838 presumptive enterococci and 209 non-enterococci) were obtained from poultry environment using the growth conditions set for isolation and characterization of enterococci. Conceivably, sampling plan covers all main areas through which enterococci disseminate into the environment from poultry. The prevalence of presumptive enterococci was high in faeces followed by air, feed and water (Table 1; Fig. 1a–b). An average count of faecal samples (n = 25) was calculated as 23 9 106 CFU g 1. The presence of Enterococcus spp. in air shows that faecal discharge is the major source of dissemination. Production of cell-bound pigments is a characteristic feature of group D Streptococcaceae (Mundt and Graham 1968). Most of these enterococci produced opaque, white and light yellow colonies; only 37% of air isolates exhibited fluorescent yellow pigmentation. Catalase-positive isolates produced predominantly white, light yellow, yellow and dark yellow colonies while orange pigmentation was observed in faecal and water isolates. Detailed analysis of 204 isolates showed six Enterococcus spp., predominantly Ent. faecium 135 (66%), Ent. faecalis 27 (13%), Ent. mundtti 8 (4%), Ent. hirae 5 (25%), Ent. gallinarum 2 (1%) and Ent. casseliflavus 1 (05%), indicating their diversity and survival in hostile environment for prolonged periods. Seventeen isolates (8%) were identified as Ent. faecalis/Ent. faecium and required further purification, seven isolates could not be characterized at species level, while two were identified as non-enterococcal streptococci (Table 1 and Fig. 2). Representative gels of identified species indicate very high reproducibility of mPCR (Fig. S1). The high prevalence of Enterococcus spp. in this study is analogous to previous reports (Oliveira et al. 2010; Champagne et al. 2011). In contrast, Poeta et al. (2006b) have found Ent. faecalis as a predominant species in poultry environment in Portugal. Analysis of interspersed repetitive DNA sequence (BOX-PCR, Jackson et al. 2006) of representative strains of six species from four different sources of poultry environment reveals that the genomic contents of Ent. faecium, Ent. faecalis and other identified species are heterogeneous, i.e. different biotypes from the same and different sample sources (Fig. S2). Enterococci are well-known opportunistic pathogens. The virulence potential of enterococci reflected by disruption of the balance commensalism, colonization, immune invasion and impairment of antibiotic action plays an important role in host with undermined defence (Poeta Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
Assessment of virulence determinants (%) Catalase – – Gelatinase 22 (275) 1 Haemolysis 8 (10) 0 Bacterial 12 (15) 0 antagonism Biofilm 34 (425) 7 formation Assessment of antibiotics resistance (%) Meropenem 65 (81) 20 Ceftriaxone 50 (625) 11 Ampicillin 22 (275) 0 Vancomycin 15 (19) 0 Oxytetracycline 70 (875) 20 Lincomycin 70 (875) 19 Clarithromycin 70 (875) 18 Erythromycin 63 (79) 12 Kanamycin 59 (74) 19 Chloramphenicol 21 (26) 5 High-level gentamicin 20 (25) 0 Ciprofloxacin 47 (59) 3
Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
(135)
(91) (86) (82) (545) (86) (23)
(91) (50)
(32)
(45)
13 4 0 0 12 13 0 3 7 0 0 5 (33)
(20) (47)
(80) (87)
(87) (27)
7 (47)
– 6 (40) 1 (7) 0
17 10 1 5 17 13 3 8 17 2 2 7
(94) (555) (55) (625) (94) (72) (17) (44) (94) (11) (11) (39)
9 (50)
– 5 (28) 6 (33) 3 (17)
Air 18
21 9 5 5 21 21 19 15 7 7 12 6
(100) (43) (24) (24) (100) (100) (90) (71) (33) (33) (57) (28)
2 (95)
2 (95) 0 0 6 (28)
1 1 0 0 1 1 0 0 0 0 0 0
0
(100) (100)
(100) (100)
1 (100) 0 0 1 (100)
Water 1
Poultry faeces 21
Water 15
Poultry faeces 80
Isolates identified n = 178 (8725%)
Poultry feed 22
Ent. faecalis n = 27 (15%)
Ent. faecium n = 135 (76%)
Total isolates n = (204)
Percentage prevalence, distribution and identification of Enterococcus spp.
1 (100) 0 0 0 1 (100) 0 0 0 0 0 0 0
0
0 1 (100) 0 0
Poultry feed 1
Table 1 Summary of the antibiotic resistance and virulence determinants of the identified Enterococcus spp.
(25) (50) (50) (50)
3 2 2 3 3 3 2 2 3 1 1 3
(75) (50) (50) (75) (75) (75) (50) (50) (75) (25) (25) (75)
2 (50)
1 2 2 2
Air 4
7 7 0 0 7 7 0 0 0 0 0 1
(14)
(100) (100)
(100) (100)
1 (14)
– 0 0 0
Water 7
1 1 0 0 1 1 1 0 1 1 0 0
0
– 0 0 0
(100) (100)
(100) (100) (100)
(100) (100)
Air 1
Ent. munditti n = 8 (5%)
1 1 0 0 1 1 0 1 0 0 1 0
0
(100)
(100)
(100) (100)
(100) (100)
– 1 (100) 0 0
Poultry faeces 1
4 2 0 0 4 3 0 0 0 0 0 1
(25)
(100) (75)
(100) (50)
1 (25)
– 0 1 (25) 1 (25)
Water 4
Ent. hirae n = 5 (3%)
2 2 1 1 2 2 2 2 2 1 1 1
(100) (100) (50) (50) (100) (100) (100) (100) (100) (50) (50) (50)
1 (50)
– 1 (50) 0 1 (50)
Air 2
Ent. gallinarum n = 2 (1%)
0 0 0 0 1 1 1 0 0 1 0 0
(100)
(100) (100) (100)
1 (100)
– 0 0 0
Poultry feed 1
Ent. casseliflavus n = 1 (06%)
S. A. Ali et al. Environmental enterococci in poultry-related environment
3
Environmental enterococci in poultry-related environment
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683
(a) 700
Number of isolates
600 500 400 300 200 200 90
100 6 0
Poultry feces/Manure
174
166
200
5
72
95
79
24
Poultry feed
Water samples
Air samples
(b) 100
87·3 76·4
Percent isolates
80
70·8 55·4
60 44·6 40
29·2 23·6
20
0
12·7
Poultry feces/Manure
Poultry feed
Water
Air
Figure 1 (a) Graphical representation of the samples collected ( ), prevalence of enterococci ( ) and isolates recovered ( ) from samples related to poultry environment randomly collected from all over Karachi city. (b) Characterization of isolates grown in BHI containing 65% NaCl at 45°C. Isolates with the ability to hydrolyse bile aesculin checked for catalase activity are shown – catalase-negative ( ) presumptive Enterococci and catalase-positive ( ) non-Enterococci.
et al. 2006b; Tyne et al. 2013). Despite limitations, in vitro assays provide useful information for establishing the actual virulence potential expressed by pathogens during the infection process, such as genotypes corresponding to virulence as well as their expression capacity, for example, in enterococci production of gelatinase (gelE), catalase (kcat), haemolysis (cylL/S), bacterial antagonism (enterocins) and biofilm formation via enterococcal surface protein (Esp), aggregation substance (Agg), etc. Virulence potential with innate or acquired resistance to antibiotics is a major predisposing factor that results in invasion of pathogen (Gilmore et al. 2002). In the present study, phenotypic assessment of virulence potential has been made using catalase, biofilm formation, enterocin and gelatinase production as well as haemolysis. Our results show that virulence potential is recurrent in presumptive enterococci and non-enterococci, but the tendency of being virulent varies accordingly 4
with the source of isolation. In general, presumptive enterococci exhibited moderate level of gelatinase production, haemolysis, bacterial antagonism and biofilm formation. The highest virulence potential was exhibited by faecal enterococci followed by water, air and feed isolates. Enterococci from feed were non-haemolytic and did not reveal bacterial antagonism. The antagonism and biofilm formation were comparatively high in faecal and air enterococci, respectively. The non-enterococci exhibited greater virulence potential in the form of high gelatinase production, haemolysis, antagonism and moderate to high biofilm formation. Highest gelatinase production and haemolysis were observed in non-enterococci isolated from faecal and air, while bacterial antagonism and biofilm formation were highest among non-enterococci from feed. High level of biofilm formation and gelatinase production among both isolates was a common trait associated with poultry environment. However, gelatinase Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
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Environmental enterococci in poultry-related environment
220 204 200 180
Number of isolates
160 135
140 120 100 80 60 40
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Species identified Figure 2 Histogram represents the prevalence of different Enterococcus spp. in the poultry and its related environment. Isolates (n = 204) were randomly selected for identification using multiplex PCR from a total of 1047 isolates to assess biodiversity. See ‘Materials and Methods’ for detail.
expression and haemolytic activity were relatively high in non-enterococci (Table 1; Fig. S3a–b). Among identified species, many isolates had multiple virulence determinants (MVD); in particular, Ent. faecium showed high potential of virulence mostly associated with faeces followed by air, water and feed, suggesting faeces as a potential reservoir of MVD strains. Biofilm, gelatinase production and haemolysis were recurrent in Ent. faecium from faeces, air, water and feed, while bacterial antagonism was found only in faecal and air isolates. The distribution of virulence determinants in Ent. faecalis and other species was not recurrent from all environmental samples, but of four Ent. faecalis from air, two were found to possess all virulence traits. Moreover, Ent. faecalis from faeces 2 (95%), water 1 (100%) and air 1 (25%) were found to produce catalase. High-level biofilm production was determined as a common attribute among the identified species (Table 1). Resistance phenotypes for three beta-lactams (meropenem, ampicillin and ceftriaxone) and one glycopeptide (vancomycin) as cell wall synthesis inhibitor, seven protein synthesis inhibitors namely chloramphenicol, oxytetracycline, lincomycin, clarithromycin, erythromycin, Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
aminoglycosides (gentamicin and kanamycin) and one DNA synthesis inhibitor carboxy-fluoroquinoline (ciprofloxacin) were determined for all isolates. High indices of multiple antibiotic-resistant (having resistance to more than two antibiotics) isolates were observed in this study. In addition to this, presence of vancomycin (6 lg ml 1)-resistant isolates and high-level-ampicillin (30 lg ml 1)-, clarithromycin (32 lg ml 1)-, erythromycin (50 lg ml 1)-, kanamycin (500 lg ml 1)- and gentamicin (512 lg ml 1)resistant enterococci and non-enterococci is also indicated (Fig. S4a–b). Chickens are more often exposed to antimicrobial agents either as therapy or as a growth promoter, which results in developing resistance in the enterococci commonly inhabiting the GIT (Diarra et al. 2010). Thus, MAR isolates should be seen as a serious threat to public health in Karachi city as the same class of antibiotic is being used in humans as well. A high number of meropenem- and ceftriaxone-resistant enterococci were observed in faeces followed by feed, air and water, while resistance to vancomycin and ampicillin was observed in air and faecal enterococci. Surprisingly, resistance to vancomycin was high in air (195%) as compared to faecal enterococci (47%) (Fig. S4a–b). 5
Environmental enterococci in poultry-related environment
Enterococci from feed and water were susceptible to vancomycin, a clear evidence that avoparcin is not being used as a growth promoter during poultry production (Kaszanyitzky et al. 2007). Apart from Ent. casseliflavus, persistent resistance to meropenem and ceftriaxone was observed among identified species. Several strains of Ent. faecium, Ent. faecalis and Ent. gallinarum isolated from faeces and air were resistant to all cell wall synthesis inhibitors included in this study, but predominant resistance to beta-lactams and glycopeptides was observed in Ent. faecium. All identified species from feed and water isolates were susceptible to vancomycin and ampicillin (Table 1). Production of low-affinity penicillin-binding proteins (PBPs) gives intrinsic resistance against beta-lactams to enterococci. Isolation of meropenem-, ceftriaxone- and ampicillin-resistant enterococci gives clear evidence of having natural resistance to these beta-lactams. Our results show that environmental enterococci exhibit different resistance profile with respect to the source of isolation and concentrations of beta-lactams used in this study. Increased susceptibility to ceftriaxone at break point (15 lg ml 1) was observed as compared to meropenem (1 lg ml 1) to which more than 70% of enterococci were resistant. In comparison, susceptibility of ampicillin (30 lg ml 1) was high among the beta-lactams as the concentration of ampicillin was double to ceftriaxone (Fig. S4a–b). Highest resistance to meropenem, ceftriaxone and ampicillin was observed in faecal and air enterococci, whereas no ampicillin-resistant isolates were recovered from feed and water. The predicted MIC of ampicillin for enterococci ranges from 10 to 16 lg ml 1, with higher concentrations, i.e. above 16 lg ml 1, corresponding to increased ampicillin resistance via PBPs over production (Kak and Chow 2002). We have noticed increased resistance to ampicillin (30 lg ml 1) in faecal and air enterococci (Table 1; Fig. S4a–b). In our study, inhibitors of protein synthesis are grouped according to ribosomal subunit targets. Low number of resistant isolates at break point value indicates effectiveness of the antibiotic. Highest resistance to oxytetracycline, lincomycin, clarithromycin, erythromycin and kanamycin was observed in presumptive enterococci, while susceptibility to chloramphenicol was observed only in enterococci from water. In contrast to kanamycin, susceptibility to gentamicin was high among the environmental enterococci (Fig. S4a–b). We have observed very high number of Ent. faecium strains, followed by Ent. faecalis and Ent. gallinarum, which were resistant to multiple protein synthesis inhibitors. In addition, high-level-gentamicin-resistant (512 lg ml 1) Ent. faecium, Ent. faecalis, Ent. hirae and Ent. gallinarum were also observed in faecal and air samples (Table 1). Our results are quite similar for resistance profile of ampicillin, tetracycline, erythromycin, chloramphenicol and high-level 6
S. A. Ali et al.
gentamicin in poultry isolates with Ent. faecium as predominant MAR of poultry origin in Portugal (Poeta et al. 2006a). Very less susceptibility to oxytetracycline in enterococci is possibly due to active efflux of drug across the cell membrane and ribosomal protection by alteration in conformation. On the other hand, limited transport of aminoglycosides across the cell membrane corresponds to low-level intrinsic resistance in enterococci. Aminoglycoside-modifying enzymes, phosphotransferases (APHs), acetyl transferases (ACCs) or nucleotidyl transferases (ANTs) are responsible for high-level-aminoglycoside resistance in enterococci (Kak and Chow 2002; Chan et al. 2008). In this study, most of the high-level-gentamicin-resistant isolates were also found to be resistant to high level of kanamycin, whereas isolates only resistant to kanamycin were susceptible to gentamicin (Table S1). Enterococci produce a distinct enzyme phosphotransferase APH (3′)-IIIa which confers high-level-kanamycin resistance, while all other aminoglycoside-modifying enzymes confer resistance to more than one aminoglycoside at a time. The isolates having resistance to kanamycin and susceptible to gentamicin strengthen the possibility of expression of phosphotransferases APH (3′)-IIIa. With the exception of chloramphenicol, enterococci showed very little response towards lincomycin, clarithromycin and erythromycin. Low-levelerythromycin resistance in enterococci (MIC 2–6 lg ml 1) is associated with an efflux protein. In our study, we have determined high level of resistance to clarithromycin (32 lg ml 1) and erythromycin (50 lg ml 1). This resistance level exempts the involvement of efflux protein. Isolates having a phenotype (MLSB) regulated by erm(B) gene in enterococci confer high-level resistance (≥32 lg ml 1) not only to macrolides but also to lincosamide. Another phenotype nucleotidyltransferase that adenylates a hydroxyl group in lincomycin also plays an important role in resistance (Kak and Chow 2002). A moderate to high number of ciprofloxacin-resistant enterococci were observed in faeces 60% followed by air 28%, water 25 and feed 6% (Fig. S4a–b). Around 59% of ciprofloxacin-resistant Ent. faecium were characterized from faeces followed by air, water and feed. Enterococcus faecalis from faeces (28%) and air (75%) were also resistant to ciprofloxacin. Contrary to the above results, Ent. faecalis from water and feed, Ent. munditti from air, Ent. hirae from faeces and Ent. casseliflavus from feed were susceptible to ciprofloxacin (Table 1). Isolates susceptible to ciprofloxacin were defined as lowlevel ciprofloxacin resistant as they exhibited MIC of 4 lg ml 1. Enrofloxacin (quinolones) is approved for use in broiler production, which might contribute towards resistance to ciprofloxacin (Martins da Costa et al. 2006). Convincingly, most of the isolates were characterized as MAR reflecting the extensive use of broad-spectrum antibiotics for disease prevention in poultry production, for Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
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example, reported recently in US conventional poultry farms (Sapkota et al. 2011). Likewise, non-enterococcal isolates also exhibit resistance to ceftriaxone and meropenem. Interestingly, a high number of ampicillin- and vancomycin-resistant non-enterococcal isolates have also been observed (Fig. S4a–b). Susceptibility to vancomycin was also recurrent in feed non-enterococci. Conversely, increased susceptibilities to meropenem and ceftriaxone in non-enterococci suggest that penicillin-binding proteins have low affinity for binding to meropenem and ceftriaxone or PBPs produced by non-enterococci are not identical with the enterococcal one. Increased level of ampicillin resistance in nonenterococci conforms with the production of PBPs with high affinity or extensively modified as compared to enterococci. Highest resistance to oxytetracycline, lincomycin, clarithromycin, erythromycin and kanamycin was also recurrent in non-enterococcal isolates. Among protein synthesis inhibitors, susceptibility to chloramphenicol was high and no resistant non-enterococci were recovered from water and feed (Fig. S4a–b). An increased resistance to kanamycin was observed in feed non-enterococci (100%) followed by air (976%), faeces (805%) and water (50%). In contrast to kanamycin, less resistance to highlevel gentamicin was observed in non-enterococci and also alike to enterococci. Moderate number of ciprofloxacin-resistant non-enterococci isolates were found in this study (Table 1; Fig. S4a–b). Cumulatively, our findings strongly recommend the need for reducing antibiotic usage, particularly for antibiotics that are being used to treat human infections, during poultry production. Materials and methods Sample collection A total of 391 samples from poultry environment (i.e. poultry faeces/manures n = 200, poultry feed n = 6, water n = 90 and air n = 95) were collected during June to October 2010 from multiple consumer shops (retailer shops), two poultry farms and surrounding environment within Karachi city. Faecal material, feed and water samples were collected aseptically in sterile glass screwcap tubes. Air was sampled as described by Berrang et al. (1995) with slight modifications; briefly, open-plate air samples were collected using petri dishes (100 mm diameter) containing freshly prepared bile aesculin azide agar (Oxoid, Hampshire, UK). Plates were placed at a height of five feet above the floor under the poultry shades and allowed to remain uncovered for 5 min for air exposure. Plates were incubated at 37°C for 24 h in aerobic incubator. All samples were carried to laboratory in an ice container within 2 h. Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
Environmental enterococci in poultry-related environment
Isolation and enumeration of Enterococcal isolates Serial dilutions 10 1–10 6 of faecal samples were made in normal saline for CFU g 1 determination. For isolation, samples were diluted 1 : 10 (w/v) and homogenized in physiological saline; 100 ll samples was inoculated and preincubated for 8–12 h at 45°C in 900 ll BHI (Oxoid) broth containing 65% NaCl to evaluate tolerance to osmotic pressure and temperature stress. A loopful of 12-h culture was used to isolate enterococci by streaking on bile aesculin azide agar plates. Colonies with brown-black zone of aesculin hydrolysis were considered as presumptive enterococci, and five morphologically distinct colonies were selected per sample (Baele et al. 2000). All enterococcal isolates were reconfirmed by Gram stain (Scharlau, Barcelona, Spain) and growth on Slanetz and Bartley Medium (Oxoid) at 37°C. Biodiversity and identification Pigmentation and colonial morphology of the isolates were observed by spot inoculation on BHI agar followed by incubation at 37°C for 24–48 h (Devriese et al. 1995). A total of 1047 isolates (838 enterococci and 209 non-enterococci) were recovered, and random selection was made for 204 presumptive enterococci (poultry faeces/manure n = 118, feed n = 28, water n = 28 and air n = 30) having diverse virulence and resistance phenotypes for multiplex identification. For complementation of these, 70 (poultry faeces/manure n = 36, feed n = 5, water n = 13 and air n = 16) were also subjected to biochemical identification using RapId STR Kits (Remel, Lenexa, KS, USA). Enterococcus faecalis ATCC 51299 (Bridier et al. 2010), Ent. casseliflavus ATCC 25788, Ent. raffinosus ATCC 49464, Ent. durans ATCC 6056, Ent. hirae ATCC 8043, Ent. faecium ATCC 6569 and indigenous Ent. faecium NA283 were taken as a reference (Oxoid). Multiplex PCR identification and BOX-PCR analysis Template DNA was prepared as described by Champagne et al. (2011). Genus (16s rDNA)- and species-specific (SodA) primers were synthesized from Operon (USA) as described recently by Jackson et al. (2012). Stock solution of primers was prepared in TE buffer (1 mol l 1 TrisHCl buffer, pH 80, containing 025 mol l 1 EDTA). mPCR was performed in seven groups of different sets of (SodA) primer along with genus-specific primers as described by Jackson et al. (2004; 2012) with slight modifications. Briefly, Master mix consists of Kapa2G Fast Hotstart ready mix (Kapa BioSystems, Wilmington, MA, USA), 15 mmol l 1 additional MgCl2, 125 ll of genus primers (16 lmol l 1), 25 and 125 ll (16 lmol l 1) SodA primers for Ent. faecalis, Ent. gallinarum, Ent. dispar, Ent. 7
Environmental enterococci in poultry-related environment
saccharolyticus and Ent. malodoratus, and for all other species; 15 ll of lysate was used as template. PCR was performed with Master cycler (ProS eppendorf, Germany) using the following conditions: initial denaturation at 95°C for 4 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C (groups 1, 2, 5 and 6) or 60°C (3, 4 and 7) for 1 min and elongation at 72°C for 1 min with final extension at 72°C for 7 min. Electrophoresis of the PCR products was performed in 2% agarose gel for 45 min at 100 V followed by visualization in UV transilluminator after staining with ethidium bromide. And 1 kb ultralow DNA ladders (Fermentas, Leon-Rot, Germany) were used as known markers. BOX-PCR analysis was ideally performed using BOXA2R primer (5′-ACG TGG TTT GAA GAG ATT TTC G-3′) as described by Jackson et al. (2006) with the only exception that Hot Start Taq Master mix kit (Qiagen, Hilden, Germany) was used for amplification. Virulence determinants Catalase activity was determined by placing 5% hydrogen peroxide (H2O2) solution on colonies grown on nutrient agar plates. Gelatinase production was evaluated on nutrient agar plates containing 04% (w/v) microbiological grade gelatin (Oxoid). Isolates were spot-inoculated for 24–48 h aerobically at 37°C (Whaley et al. 1982). The zones of gelatin hydrolysis were developed by saturated ammonium sulfate solution (Merck, Darmstadt, Germany). For haemolysis (cytolysin production), all isolates were grown on 5% human blood agar plates (Oxoid) at 37°C in CO2 incubator (Healforce, Taipei, Taiwan), as described by Clyne et al. (1988). The greenish and clear zone around the colonies were recorded as a- and b-haemolysis, respectively. No zone was considered as no or c-haemolysis (Diarra et al. 2010). Bacterial antagonism (bacteriocinogenesis) was performed by spot over lawn method (Del Campo et al. 2001); 150 ll of an overnight culture of indicator or sensitive strain (Ent. faecium NA283) was seeded to 1% BHI agar plates. After solidification, 3 ll of test isolates was spotted and incubated overnight at 37°C for 24–48 h to observe the zone of inhibition around the colonies. Biofilm formation was detected by culturing the isolates at 37°C in trypticase-soy broth (TSB) with 05% glucose. Overnight culture was diluted 1 : 40 in fresh TSB–05% glucose; 200 ll of the diluted culture was added to flatbottomed polystyrene microtitre plates in triplicate and incubated for 48 h at 37°C. Plates were washed gently three times with distilled water using automated plate washer (Columbus TECAN, M€annedorf, Austria). After drying the plates at room temperature for 1 h, the adherent biofilm was stained with 01% safranin (Scharlau) for 20 min at 8
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room temperature. Mean OD values were determined at 490 nm in microtitre plate reader (Sunrise TECAN, M€annedorf, Austria). TSB–05% glucose blank and biofilm-producing Ent. faecalis ATCC 51299 strain were taken as negative and positive controls, respectively. OD values ≥0081 and ≥02 were considered as moderate and high biofilm producers, respectively. Values below 0081 were categorized into low or non-biofilm producers. Collectively, values from 0081 to 02 or more were considered as production of biofilm (Giridhara Upadhyaya et al. 2010). Antibiotics resistance Resistance phenotypes were determined on VRE agar base (Oxoid) by incorporating antibiotic supplements as per (NCCLS, 1997) guidelines for break points classification as resistant to meropenem (1 lg ml 1), vancomycin (6 lg ml 1), ceftriaxone (15 lg ml 1), ampicillin (30 lg ml 1), chloramphenicol (32 lg ml 1), oxytetracycline (16 lg ml 1), lincomycin (4 lg ml 1), clarithromycin (32 lg ml 1), erythromycin (50 lg ml 1), kanamycin (500 lg ml 1), gentamicin (512 lg ml 1) and ciprofloxacin (4 lg ml 1) (Oxoid). All isolates were grown for 24–48 h at 37°C, and black zone of aesculin hydrolysis was considered as resistant enterococci (Furtula et al.2013). Enterococcus faecalis ATCC 51299 strain (low-level vancomycin resistant) and Ent. faecium NA283 were also taken as positive and negative controls, respectively. Acknowledgements This project was financially supported by Higher Education Commission (HEC), Islamabad, Pakistan, to SA Ali (HEC No. 20-1339/R&D/09). Conflict of interest Authors declare no conflict of interest. Furthermore, there is no human and/or animal studies involved in this purely academic research. References Baele, M., Baele, P., Vaneechoutte, M., Storms, V., Butaye, P., Devriese, L.A., Verschraegen, G., Gillis, M. et al. (2000) Application of tRNA intergenic spacer PCR for identification of Enterococcus species. J Clin Microbiol 38, 4201–4207. Berrang, M.E., Cox, N.A. and Bailey, J.S. (1995) Measuring air-borne microbial contamination of broiler hatching cabinets. J Appl Poult Res 4, 83–87. Bridier, A., Dubois-Brissonnet, F., Boubetra, A., Thomas, V. and Briandet, R. (2010) The biofilm architecture of sixty
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opportunistic pathogens deciphered using a high throughput CLSM method. J Microbiol Methods 82, 64–70. Champagne, J., Diarra, M.S., Rempel, H., Topp, E., Greer, C.W., Harel, J. and Masson, L. (2011) Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry. Appl Environ Microbiol 8, 2625–2633. Chan, Y.Y., Abd Nasir, M.H., Yahaya, M.A., Salleh, N.M., Md Dan, A.D., Musa, A.M. and Ravichandran, M. (2008) Low prevalence of vancomycin and bifunctional aminoglycoside-resistant enterococci isolated from poultry farms in Malaysia. Int J Food Microbiol 122, 221–226. Clyne, M., De Azavedo, J., Carlson, E. and Arbunthnott, J. (1988) Production of gamma-haemolysin and lack of production of alpha-haemolysin by Staphylococcus aureus strains associated with toxic shock syndrome. J Clin Microbiol 26, 535–539. De Baere, T., Claeys, G., Verschraegen, G., Devriese, L.A., Baele, M., Van Vlem, B., Vanholder, R., Dequidt, C. et al. (2000) Continuous peritoneal dialysis peritonitis due to Enterococcus cecorum. J Clin Microbiol 38, 3511–3512. De Niederh€ausern, S., Bondi, M., Anacarso, I., Iseppi, R., Sabia, C., Bitonte, F. and Messi, P. (2013) Antibiotics and heavy metals resistance and other biological characters in enterococci isolated from surface water of Monte Cotugno Lake (Italy). J Environ Sci Health A Tox Hazard Subst Environ Eng 48, 939–946. Del Campo, R., Tenorio, C., Jimenez-Dıaz, R., Rubio, C., G omez-Lus, R., Baquero, F. and Torres, C. (2001) Bacteriocin production in vancomycin-resistant and vancomycin-susceptible Enterococcus isolates of different origins. Antimicrob Agents Chemother 45, 905–912. Devriese, L.A., Pot, B., Van Damme, L., Kersters, K. and Haesebrouck, F. (1995) Identification of Enterococcus species isolated from foods of animal origin. Int J Food Microbiol 26, 187–197. Diarra, M.S., Rempel, H., Champagne, J., Masson, L., Pritchard, J. and Topp, E. (2010) Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens. Appl Environ Microbiol 76, 8033–8043. Expert Panel on Antibiotic Resistance. (2005) A review of the impact of the use of antimicrobials in animals and plants on the development of antimicrobial resistance in human bacterial pathogens. NZFSA Wellington (available on-line http://www.foodsafety.govt.nz). Foulquie Moreno, M.R., Sarantinopoulos, P., Tsakalidou, E. and De Vuyst, L. (2006) The role and application of enterococci in food and health. Int J Food Microbiol 106, 1–24. Frankenberg, L., Brugna, M. and Hederstedt, L. (2002) Enterococcus faecalis heme-dependent catalase. J Bacteriol 184, 6351–6356. Furtula, V., Jackson, C.R., Farrell, E.G., Barrett, J.B., Hiott, L.M. and Chambers, P.A. (2013) Antimicrobial resistance
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in Enterococcus spp. isolated from environmental samples in an area of intensive poultry production. Int J Environ Res Public Health 10, 1020–1036. GALLUP PAKISTAN. (2008-9) Poll findings on meat consumption survey. Press Release by Gilani Research Foundation (available on-line http://www.gallup.com.pk). Giard, J.C., Laplace, J.M., Rince, A., Pichereau, V., Benachour, A., Leboeuf, C., Flahaut, S., Auffray, Y. et al. (2001) The stress proteome of Enterococcus faecalis. Electrophoresis 22, 2947–2954. Gilmore, M.S., Coburn, P.S., Nallapareddy, S.R. and Murray, B.E. (2002) Enterococcal Virulence. In The Enterococci: Pathogenesis, Molecular Biology and Antibiotic Resistance Gilmore MS ed. Clewell, D.B., Courvalin, P., Dunny, G.M., Murray, B.E. and Rice, L.B. pp. 301–313. Washington: ASM Press. Giridhara Upadhyaya, P.M., Umapathy, B.L. and Ravikumar, K.L. (2010) comparative study for the presence of enterococcal virulence factors gelatinase, hemolysin and biofilm among clinical and commensal isolates of Enterococcus faecalis. J Lab Physicians 2, 100–104. Greub, G., Devriese, L.A., Pot, B., Dominguez, J. and Bille, J. (1997) Enterococcus cecorum septicemia in a malnourished adult patient. Eur J Clin Microbiol Infect Dis 16, 594–598. Jackson, C.R., Fedorka Cray, P.J. and Barrett, J.B. (2004) Use of a genus and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol 42, 3558– 3565. Jackson, C.R., Debnam, A.L., Avellaneda, G.E., Barrett, J.B. and Hofacre, C.L. (2006) Effect of subtherapeutic antimicrobials on genetic diversity of Enterococcus faecium from chickens. Avian Dis 50, 115–119. Jackson, C.R., Furtula, V., Farrell, E.G., Barrett, J.B., Hiott, L.M. and Chambers, P. (2012) A comparison of BOX-PCR and pulsed-field gel electrophoresis to determine genetic relatedness of enterococci from different environments. Microb Ecol 64, 378–387. Kak, V. and Chow, W.J. (2002) Acquired Antibiotic resistance in Enterococci. In The Enterococci: Pathogenesis, Molecular Biology and Antibiotic Resistance Gilmore MS ed. Clewell, D.B., Courvalin, P., Dunny, G.M., Murray, B.E. and Rice, L.B. pp. 355–372. Washington: ASM Press. Kakinuma, Y. (1998) Inorganic cation transport and energy transduction in Enterococcus hirae and other Streptococci. Microbiol Mol Biol Rev 62, 1021–1045. Kapoor, L., Randhawa, V.S. and Deb, M. (2005) Antimicrobial resistance of enterococcal blood isolates at a pediatric care hospital in India. Jpn J Infect Dis 58, 101–103. Kaszanyitzky, E.J., Tenk, M., Ghidan, A., Fehervari, G.Y. and Papp, M. (2007) Antimicrobial susceptibility of Enterococci strains isolated from slaughter animals on the data of Hungary resistance monitoring system from 2001 to 2004. Int J Food Microbiol 115, 119–123. Martins da Costa, P.M., Vaz-Pires, P.M. and Bernardo, F.M. (2006) Antimicrobial resistance of Enterococcus spp.
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Isolated from waste water and sludge of poultry slaughter houses. J Environ Sci Health B 41, 1393–1403. Moraes, P.M., Perin, L.M., Todorov, S.D., Silva, A. Jr, Franco, B.D. and Nero, L.A. (2012) Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese. J Appl Microbiol 113, 318–328. Mundt, J.O. and Graham, W.F. (1968) Streptococcus faecium var.casselliflavus, nov. var. J Bacteriol 95, 2005–2009. National Committee for Clinical Laboratory Standards. (1997) Specialty collection: Susceptibility Testing. SC21-L.M7-A4. NCCLS, PA. 3. Wayne. Oliveira, M., Santos, V., Fernandes, A., Bernardo, F. and Vilela, C.L. (2010) Antimicrobial resistance and in vitro biofilmforming ability of enterococci from intensive and extensive farming broilers. Poult Sci 89, 1065–1069. Poeta, P., Costa, D., Rodrigues, J. and Torres, C. (2006a) Antimicrobial resistance and mechanisms implicated in fecal Enterococci from healthy humans, poultry and pets in Portugal. Int J Antimicrob Agents 27, 131–137. Poeta, P., Costa, D., Klibi, N., Rodrigues, J. and Torres, C. (2006b) Phenotypic and genotypic study of gelatinase and beta-haemolysis activities in faecal enterococci of poultry in Portugal. J Vet Med B Infect Dis Vet Public Health 53, 203–208. Poulsen, L.L., Bisgaard, M., Son, N.T., Trung, N.V., An, H.M. and Dalsgaard, A. (2012) Enterococcus faecalis clones in poultry and in humans with urinary tract infections, Vietnam. Emerg Infect Dis 18, 1096–1100. Sapkota, A.R., Hulet, R.M., Zhang, G., McDermott, P., Kinney, E.L., Schwab, K.J. and Joseph, S.W. (2011) Lower prevalence of antibiotic-resistant enterococci on US conventional poultry farms that transitioned to organic practices. Environ Health Perspect 119, 1622–1628.
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Stalker, M.J., Brash, M.L., Weisz, A., Ouckama, R.M. and Slavic, D. (2010) Arthritis and osteomyelitis associated with Enterococcus cecorum infection in broiler and broiler breeder chickens in Ontario, Canada. J Vet Diagn Invest 22, 643–645. Tyne, D.V., Martin, J.M. and Gilmore, M.S. (2013) Structure, function and biology of the Enterococcus faecalis cytolysin. Toxins 5, 895–911. Velkers, F.C., van de Graaf-Bloois, L., Wagenaar, J.A., Westendorp, S.T., van Bergen, M.A., Dwars, R.M. and Landman, W.J.M. (2011) Enterococcus hirae-associated endocarditis outbreaks in broiler flocks: clinical and pathological characteristics and molecular epidemiology. Vet Q 31, 3–17. Whaley, D.N., Dowell, J.R., Wanderlinder, L.M. and Lombard, G.L. (1982) Gelatin agar medium for detecting gelatinase production by anaerobic bacteria. J Clin Microbiol 16, 224–229.
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1 Identification of Enterococcal isolates by Multiplex PCR using 2% agarose gel analysis. Fig. S2 Molecular assessment of inter-strain variation established by BOX PCR using 2% agarose gel analysis. Fig. S3 Phenotypic assessment of virulence determinants. Fig. S4 Percent prevalence of resistance against three different classes of antibiotics. Table S1. Summary of high level kanamycin, gentamicin and bi-functional aminoglycosides resistant presumptive enterococci and non-enterococcal isolates.
Letters in Applied Microbiology © 2013 The Society for Applied Microbiology
