Materials and Methods
Bacterial strains. Eight S. marcescens isolates (MPU Si1 – MPU Si8) were used in this study. They were isolated from dead caterpillars of Spodoptera exigua cultured in the laboratory culture line in September 2009. In order to determine the clonal relationship of isolates, thirteen additional strains were included in the study as a comparative samples: S. marcescens strain MPU Si9 obtained from a dead larva of Agrotis segetum reared in the same laboratory three months earlier, eight S. marcescens strains (MPU S11, MPU S14, MPU S15, MPU S26, MPU S32, MPU S40, MPU S41, MPU S47) isolated from clinical specimens, one S. odorifera (MPU Se128/7) cultured from sewage, one S. ficaria (MPU S49), one strain of Enterobacter cloacae (MPU E39a) from clinical specimens, and Pseudomonas aeruginosa (MPU Pb5/4) from the intestinal tract of a wild boar.
Bacteria isolation and identification. Dead caterpillars were swabbed with 90% ethanol, homogenized in BHI medium (Difco) and incubated at 30°C for 24h. Then, the bacterial suspension was spread on BHI agar medium and after 24-hour incubation, the bacteria were characterized based on the morphology of colonies. Identification of bacteria was performed by API 20E tests according to the manufacturer’s instruction (bioMérieux, France).
Clonal relationship. Bacterial typing was conducted by REP-PCR with primers REP1I and REP2I for repetitive extragenic palindromic sequences (Moura et al. 2007). One bacterial colony was suspended in 25 μl of sterile distilled water and heated at 98°C for 5 min. Two microliters of DNA were added to PCR mixture containing PCR buffer with NH4(SO4)2, 0.5 μM of each primer (Oligo.pl, Poland), 200 μM of dNTP mix, 2.5 mM of MgCl2, and 0.5 U of Allegro DNA polymerase (Novazym, Poland). PCR amplification involved an initial denaturation at 94°C for 3 min followed by 35 cycles at 94°C for 1 min, 40°C for 1 min, 72°C for 90, and a final extension at 72°C for 8 min.
Amplification products were electrophoresed in 1.5% NOVA Mini agarose gel (Novazym, Poland). The similarity of bacteria DNA profiles was estimated with Dice coefficient and a dendrogram was created by the unweighted pair group method with average linkages (UPGMA).
Pathogenicity of S. marcescens strains for insects. The pathogenicity of S. marcescens strains against S. exigua caterpillars was determined. Insects originated from laboratory culture line reared at 26°C, 40–60% relative humidity, and a 16:8 (light: dark) period in the Department of Microbiology, Adam Mickiewicz University, Poznań.
The bioassay on insects was conducted according to Jeong et al. (2010), Mohan et al. (2011), and Sikorowski et al. (2001). S. marcescens isolates were grown in nutrient agar (Biocorp, Poland) at 30°C for 24 h. One bacterial colony was suspended in 1 ml of 0.85% NaCl. Five different volumes: 2.5 μl, 5 μl, 10 μl, 20 μl, 40 μl of the suspension were spread on pieces (5 mm diameter, 3 mm hight) of semisynthetic diet for S. exigua rearing as described by McGuire et al. (1997). The pieces of diet were placed separately in transparent polystyrene multi-well plates. Caterpillars in L1 instar were placed onto the diet – one larva per one well with one diet piece. Each S. marcescens CFU number was tested against 30 larvae (three replications with 10 insects each). Simultaneously, the number of CFU per milliliter was determined by the spread plate method. The volume of 100 μl of a bacterial colony suspension in 1 ml of 0.85% NaCl, and additionally the volume of 100 μl of five dilutions of the suspension (10–1, 10–2, 10–3, 10–4, and 10–5) were spread on BHI agar (each dilution on two plates with medium). After the overnight incubation at 37°C, the colonies were counted and the number of CFU per milliliter was calculated. As we mentioned above different volumes of the suspension were spread on pieces (5 mm diameter, 3 mm height) of semisynthetic diet for insect rearing. The pieces had a shape of a cylinder and the formula for the surface area is πr2. Knowing the value of the surface area in cm2, the volume of bacterial suspension spread on the medium, and the number of CFU per milliliter, we calculated CFU/cm2.
As a positive control, a strain of Bacillus thuringiensis subsp. kurstaki HD1 from biopesticide Foray was used. Foray contains bacterial endospores (spores) and protein crystals and is dedicated for plant protection against lepidopteran pests. The B. thuringiensis was cultured in medium developed for bacteria sporulation by Lecadet and Dedonder (1971). Spores and crystals were applied to insects in the same way as S. marcescens. As a negative control, 0.85% NaCl was used.
Infected insects were reared at 26°C, 40–60% relative humidity, and a 16:8 (light: dark) period. The number of dead insects was estimated after 7 days. The LC50 [S. marcescens CFU per 1 cm2 of insect diet surface] against insects was calculated by a probit analysis according to Finney, based on the mortality (%) in the control sample, by using BioStat ver. 5.8.4.3 software (AnalystSoft Inc.).
Cytotoxic activity to epithelial cells by MTT assay. African monkey kidney (Vero) cells were cultured in Eagle Minimum Essential Medium (EMEM, Sigma) with 5% fetal calf serum (FCS, Sigma) containing 2 mM glutamine, 50 IU of penicillin per milliliter, streptomycin (100 μg/ml) and nystatin (1 mg/ml). The cells were seeded (1 × 104 per well) and incubated at 37°C in an atmosphere with 5% CO2.
The strains were cultured in Luria-Bertani medium (LB, Difco) at 37°C for 24 h with shaking at 300 rpm. The supernatants were centrifuged at 3000 × g for 30 min and sterilized through 0.22 μm-pore size membrane filters Millex-GV (Millipore) (Krzymińska et al. 2010).
The epithelial cell monolayer was incubated with bacterial culture filtrates for 24 h at 37°C. As a negative control, the cells were infected with a nonpathogenic E. coli K-12C600 supernatant. The cytotoxicity was assessed quantitatively by monitoring the mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) (Krzymińska et al. 2009). The data were presented as means ± standard deviation (SD) from two independent experiments performed in duplicate.
Siderophore production.Serratia sp. strains were verified for the production of siderophores by chrome azurol S (CAS) assay (Schwyn and Neilands 1987). The type of the siderophore excreted was identified in cross-feeding assays with the following indicators: enterobactin indicator – Salmonella typhimurium TA 2700, aerobactin and rhodotorulic acid indicator – Escherichia coli LG 1522, rhizoferrin and α-keto acids indicator – Morganella morganii SBK 3 (Reissbrodt and Rabsch 1988), and yersiniabactin indicator – Yersinia enterocolitica 5030 (Haag et al. 1993). Additionally, the presence of siderophore receptors for yersiniabactin, aerobactin and glycosylated enterobactin – salmochelin, encoded respectively by the fyuA, iutA and iroN genes, was assessed by PCR. All PCR reagents were purchased from Novazym (Poland). PCR amplification conditions and sequences of primers have been previously published (Karch et al. 1999; Johnson et al. 2000; Johnson and Stell 2000). The PCR products were separated in 1.5% agarose gel. All experiments were performed in triplicate.
Antimicrobial susceptibility. The susceptibility of the isolates to 20 antibiotics representing nine classes was determined according to the standard disk diffusion method recommended by The European Committee on Antimicrobial Susceptibility Testing (EUCAST 2014). The antimicrobials comprised: amikacin (30 μg), tobramycin (10 μg), netilmicin (10 μg), gentamicin (10 μg), ticarcillin (75 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), cefotaxime (5 μg), ceftaroline (5 μg), ceftazidime (10 μg), cefoperazone (10 μg), cefepime (30 μg), co-trimoxazole (25 μg), trimethoprim (5 μg), sulfamethoxazole (200 μg), piperacillin (30 μg), piperacillin/tazobactam (30–60 μg), aztreonam (30 μg), imipenem (10 μg), and meropenem (10 μg). The production of extended spectrum β-lactamases (ESBL) was determined in the double-disc synergy test. All antibiotic discs were provided by Oxoid. Amplifications of three genes conferring resistance to sulfonamides (sul1, sul2, sul3), were conducted in a 25-μl volume with PCR buffer with NH4(SO4)2, 0.5 μM of each primer (Oligo.pl), 200 μM of dNTP mix, 2.5 mM of MgCl2, 0.5 U of DreamTaq polymerase (Thermo Scientific), and 200 ng of genomic DNA. The PCR conditions consisted of initial denaturation at 94°C, 3 min, followed by 35 cycles at 94°C for 45 s, annealing (varied; 46–60°C) for 45 s, 72°C for 90 s, with a final extension at 72°C for 7 min (Pei et al. 2006).
Presence of integrons. The integron integrase genes were detected by multiplex PCR with primers targeting three classes of the integrase genes intI1, intI2 and intI3 (Dillon et al. 2005). PCR amplifications were performed in a 25-μl volume with 2.5 μl of 10 × PCR buffer with NH4(SO4)2, 0.25 μM of each primer, 100 μM of dNTP mix, 2.5 mM of MgCl2, 1 U of DreamTaq polymerase (Thermo Scientific), and 200 ng of genomic DNA. Amplification involved an initial denaturation (94°C, 5 min) followed by 30 cycles of denaturation (94°C, 1 min), annealing (59°C, 1 min) and extension (72°C, 1 min), with a final extension step (72°C, 8 min).
