Detection of antibiotic resistance, virulence gene determinants and biofilm formation in Aeromonas species isolated from cattle Isoken H. Igbinosa, Etinosa O. Igbinosa & Anthony I. Okoh
Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-015-4934-4
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Author's personal copy Environ Sci Pollut Res DOI 10.1007/s11356-015-4934-4
RESEARCH ARTICLE
Detection of antibiotic resistance, virulence gene determinants and biofilm formation in Aeromonas species isolated from cattle Isoken H. Igbinosa 1,2 & Etinosa O. Igbinosa 1,3 & Anthony I. Okoh 1
Received: 3 January 2015 / Accepted: 22 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract This study aimed to assess the antibiogram of Aeromonas strains recovered from cattle faeces and the potential pathogenic status of the isolates. The antibiogram of the Aeromonas isolates demonstrated total resistance to clindamycin oxacillin, trimethoprim, novobiocin and ticarcillin. However, Aeromonas strains were sensitive to cefotaxime, oxytetracycline and tobramycin. The Aeromonas strains from Lovedale and Fort Cox farms were found to possess some virulence genes. The percentage distribution was aer 71.4 %, ast 35.7 %, fla 60.7 %, lip 35.7 % and hlyA 25 % for Lovedale farm and aer 63.1 %, alt 10.5 %, ast 55.2 %, fla 78.9 %, lip 21 % and hlyA 35.9 % for Fort Cox farm. Class 1 integron was present in 27 % of Aeromonas isolates; the blaTEM gene was present in 34.8 %, while the blaP1 class A βlactamase gene was detected in 12.1 % of the isolates. Approximately 86 % of the isolates formed a biofilm on microtitre plates. The presence of multiple antibiotic resistance and virulence genes in Aeromonas isolates from cattle faeces
Responsible editor: Robert Duran * Isoken H. Igbinosa
[email protected] Etinosa O. Igbinosa
[email protected] Anthony I. Okoh
[email protected] 1
SA-MRC Microbial Water Quality Monitoring Centre, University of Fort Hare, Alice, South Africa
2
Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria, Private Bag X323, Pretoria 0001, South Africa
3
Department of Microbiology, Faculty of Life Sciences, University of Benin, Private Mail Bag 1154, Benin 300001, Nigeria
reveals the pathogenic and infectious importance of these isolates and is of great significance to public health. The possession of a biofilm-forming capability by such isolates may lead to difficulty during the management of infection related to Aeromonas species. Keywords Antibiotic resistance . Integron . blaTEM . Biofilm . Virulence gene
Introduction Aeromonas species are potential food-poisoning agents; some species of Aeromonas are psychrotrophic in nature and have been associated with the spoilage of animal meat including chicken and beef (Majeed et al. 1989). Aeromonas species produce toxins at low temperature when growth conditions become suitable (Majeed et al. 1989; Krovacek et al. 1991). Risk factors associated with this disease in humans are related to the consumption of contaminated food and water as well as direct contact with contaminated animal. The detection of motile aeromonads in the faeces of cattle has been documented (Gray and Stickler 1989; Ceylan et al. 2009). The presence of Aeromonas species in commercially obtained meats and other foods of an animal source is evident in the literature (Majeed et al. 1989; Rossi Júnior et al. 2006). The presence of Aeromonas species in cow faeces could pose a possible public health challenge for individuals who might associate or come in contact with infected animals. Contaminated or infected animals can play a vital role in the transmission of aeromonads from animals or food to humans, and animal faeces appear to be the main channel of contamination of foods (Jindal et al. 1993; Ceylan et al. 2003). Antibiotic resistance among enteric pathogens is a serious problem facing developing countries where there is a high frequency of gastroenteric illnesses. Antibiotic resistance is
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relevant in pathogenic Aeromonas species in which, besides the classical resistance to β-lactamase antibiotics, multipleantibiotic resistance has been frequently identified (Kampfer et al. 1999; Vila et al. 2002). In addition, bacteria of this genus can receive and transfer antibiotic resistance genes, further increasing the threat from resistant bacterial infections (Marchandin et al. 2003; Zanella et al. 2012). Among bacteria, Aeromonas species harbour different β-lactamase genes, including cepH, cphA and ampH (Avison et al. 2004). Aeromonas species isolated from a hospital setting in Taiwan have been reported to possess a different extended spectrum of β-lactamases, including blaPER, blaTEM, blaSHV, blaCTX-M, blaCphA and the AmpC β-lactamase gene (Wu et al. 2011). Additionally, Aeromonas spp. isolated from environmental sources in Brazil demonstrated a high frequency of resistance to β-lactam antibiotics (Scoaris et al. 2008). The virulence status of a microorganism determines the extent of harmfulness and the capability of the microorganism to cause disease. Virulence factors in Aeromonas are associated with the establishment of infection and are thought to be involved in the development of disease (Nawaz et al. 2010; Nagar et al. 2011). Aeromonas causes disease in both humans and animals (Dashe et al, 2013; Igbinosa 2014). The presence of Aeromonas in the faeces of animals could be symptomatic or asymptomatic. Colonized animals could also be a source of infection for humans through the faecal-oral route. Extracellular proteins released by Aeromonas, including aerolysin, elastase, nuclease, lipase and protease (Pemberton et al. 1997), are known as virulence factors that cause disease in humans (Nam and Joh 2007) and animals. Additionally, lateral flagella are crucial for swimming, swarming motility and biofilm formation (Gavin et al. 2002; Kirov et al. 2004) and represent a potential colonization factor. Virulence in Aeromonas is multifactorial and not yet fully understood. However, the isolation and sequencing of genes encoding these virulence factors allow the detection of signature regions of these genes and the evaluation of their presence in Aeromonas isolates (Chacón 2003; Zanella et al. 2012). Continual surveillance is essential to prevent infections associated with Aeromonas. The formation of a biofilm is a significant phase in the pathogenicity of microorganisms (Bell 2001). In addition, biofilm establishment on inanimate surfaces or host tissue inactivates the efficacy of antibiotics, guards against host defence mechanisms and enhances bacterial cell communication resulting in the expression of virulence factors (Leonard et al. 2000), thereby providing a favourable niche and environment for microorganism acceleration of disease outbreaks (Karunasagar et al. 1996; Coquet et al. 2002; Basson et al. 2007). Conventionally, the study of biofilms has shown that the structural surfaces of cells, such as pilli, flagella, outer membrane proteins (OMPs) and extra-polymeric substances (EPS), function by allowing bacteria to initially stick to a surface and then form biofilms (O’Toole et al. 2000; Basson
et al. 2007). Many biofilm-associated microorganisms pose a risk to animal and human health by harbouring other pathogenic or toxin-producing microorganisms (Riley et al. 2011). In view of this information, this study aimed to assess the incidence of Aeromonas spp. in the faeces of cattle, examine their antibiotic profile and possession of some resistant gene determinants and evaluate their pathogenic status by examining their virulence potential and ability to form biofilms.
Materials and methods Collection of samples, isolation and identification of Aeromonas Cow faecal samples were collected from the ground from Lovedale and Fort Cox farms in the Eastern Cape Province of South Africa. The samples were collected at three different times, and 60 samples were collected from both farms. The samples were processed following standard culture-based methods. Five grams (5.0 g) of faecal sample was inoculated into 100 mL of tryptone soy broth (Merck, SA) and incubated in a shaker for 24 h at 37 °C; afterwards, the broth culture was streaked on several Glutamate Phenol Red (Merck, SA) agar plates for the isolation of Aeromonas spp. Phenotypically, yellow colonies were picked as presumptive Aeromonas species and purified. Aeromonas were identified based on morphological, cultural and biochemical characteristics (Quinn et al. 2002; Igbinosa and Okoh 2012). The isolates were confirmed by examining Gram staining characteristics and performing oxidase, catalase and biochemical reactions. The confirmation of the isolates was performed using an API 20NE kit (Biomerieux, Marcy-l’Etoile, France). The tests were conducted by suspending two to three colonies of test bacteria in normal saline, and the suspension was adjusted to 0.5 MacFarland standard. The suspension was then used as the inoculum in the experiment. The experiment was conducted according to the users’ guide of the API 20NE kit. The strips were incubated at 37 °C for 24–48 h, and the results were read and interpreted with the aid of the Analytical Profile Index (API) database (V4.1) and apiwebTM identification software. Antimicrobial sensitivity testing The isolates were exposed to antibiotic susceptibility testing using the disc diffusion method, as recommended by the Clinical and Laboratory Standards Institute CLSI (2006), on Mueller Hinton agar (Oxoid, Basingstoke, UK). Susceptibility testing was conducted using the following antibiotic discs: amoxicillin (30 μg), ampicillin-sulbactam (20 μg), aztreonam (30 μg), cefotaxime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), erythromycin (15 μg), gentamicin (10 μg), imipenem (10 μg), kanamycin (30 μg),
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nalidixic acid (30 μg), neomycin (30 μg), nitrofurantoin (300 μg), novobiocin (5 μg), oxacillin (1 μg), oxytetracycline (30 μg), penicillin (10 μg), polymyxin B (300 units), streptomycin (10 μg), tetracycline (10 μg), ticarcillin (75 μg), tobramycin (10 μg) and trimethoprim (5 μg). All of the discs were obtained from Mast Diagnostics, Merseyside, UK. First, pure isolates were grown on nutrient agar plates for 18 h, and thereafter, three to five colonies were suspended in normal physiological saline to obtain a turbidity of 0.5 McFarland standards. In addition, the isolate suspension was smeared onto Muller Hinton agar plates using a sterile cotton swab. The plates were allowed to dry before impregnating the antibiotic discs. The plates were then incubated at 37 °C for 24 h, after which, zones of inhibition were measured and interpreted according to the CLSI recommendation (2006). Molecular detection of genes coding for antibiotic resistance and virulence genes Polymerase chain reaction (PCR) was used to detect antibiotic resistance genes in the Aeromonas species using the specific primer pairs for pse1 (pse-F ACC GTA TTG AGC CTG ATT TA pse-R ATT GAA GCC TGT GTT TGA GC) and blaTEM (5′-AGGAAGAGTATGATTCAACA-3′ and 5′-CTCGTCGT TTGGTATGGC-3′). The cycling conditions (Bio-Rad My Cycler™ Thermal Cycler) were as follows: pse1 - PSE-1/ CARB-2 (blaP1 class A β-lactamase) (initial denaturation at 96 °C for 5 min, followed by 30 cycles of denaturation at 96 °C for 30 s, annealing at 60 °C and a single extension of 5 min at 72 °C); blaTEM (3 min at 93 °C, 40 cycles of 1 min at 93 °C, 1 min at 55 °C, 1 min at 72 °C and, finally, 7 min at 72 °C) (Igbinosa and Okoh 2012). The set of primers 5′-GGC ATC CAA GCA GCA AG-3′ and 5′-GGC ATC CAA GCA GCA AG-3′, and 5′-CGG GAT CCC CGG CAT GCA CGA TTT GTA-3′ and 5′-GAT GCC ATC GCA AGT ACG AG-3′ was used for the detection of the presence of class 1 and class 2 integrons, respectively. The PCR conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 56 °C for 1 min, extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min (Nawaz et al. 2010). The presence of putative virulence genes coding for aerolysin (aer), heat-labile cytotonic enterotoxin (alt), flagellin (fla), heat-stable cytotonic enterotoxin (ast), lipase (lip) and cytotoxin (hlyA) was determined by PCR using primers and conditions previously published in Igbinosa and Okoh (2013). Detection and quantification of biofilm formation potentials Aeromonas isolates were grown overnight in tryptone soy broth (TSB) at 37 °C and centrifuged for 120 s at 12,000×g.
The pelleted cells were washed and resolubilized in phosphate-buffered saline (PBS, pH 7.2) and adjusted to 0.5 McFarland standards (Basson et al. 2007). Bacterial adherence to an abiotic surface was determined by inoculating wells of sterile 96-well polystyrene microtitre plates with 180 μL of tryptone soy broth and 20 μL of standardized cell suspensions (Stepanović et al. 2000; Igbinosa et al. 2013). The negative control wells contained only TSB broth or PBS and were included in wells as negative controls while Aeromonas hydrophila ATCC 7966 was included as a positive control. The microtitre plates were incubated at 37 °C for 24 h. The wells were carefully aspirated and washed three times with sterile PBS at pH 7.2, and the remaining PBS was discarded. After air drying, the wells were stained with 200 μL of 1 % crystal violet for 30 min. The wells were washed with distilled water to minimize the surplus stain, and the plates were allowed to air dry at normal atmospheric temperature. The crystal violet dye bound to adherent cells was resolubilized with 150 μL of absolute ethanol. The optical density (OD) value of all wells was recorded at 570 nm with the aid of a microtitre plate reader (SynergyMx BiotekR USA). The assays were conducted in triplicate, and the average results obtained (Stepanović et al. 2000; Igbinosa et al. 2013). The formation of a biofilm was categorized as a non-producer or a weak, moderate or strong producer. The average optical density (OD) of each duplicate result was determined, including those of the positive and negative controls. The isolates were categorized as non-biofilm (ODi
