Pseudomonas and Pedobacter isolates
from King George Island inhibited the growth
of foodborne pathogens
Clemente Michael Vui Ling WONG 1, Heng Keat TAM 1, Siti Aisyah ALIAS 2, Marcelo GONZÁLEZ 3, Gerardo GONZÁLEZ−ROCHA4 1 Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia 2 Institute of Biological Science, Faculty of Science, Universiti Malaya, 3 Departamento Científico, Instituto Antártico Chileno, Plaza Muńoz Gamero 1055, Punta Arenas, Chile 4 Departamento Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, K e y w o r d s : Antarctic, antimicrobial, foodborne pathogen, 16S rDNA sequence.
Abstract: This report describes the isolation and characterization of bacterial isolates that
produce anti−microbial compounds from one of the South Shetland Islands, King George Is−
land, Antarctica. Of a total 2465 bacterial isolates recovered from the soil samples, six
(BG5, MTC3, WEK1, WEA1, MA2 and CG21) demonstrated inhibitory effects on the
growth of one or more Gram−negative or Gram−positive indicator foodborne pathogens (i.e.
Escherichia coli
0157:H7, Salmonella spp., Klebsiella pneumoniae, Enterobacter cloacae,
Vibrio parahaemolyticus and Bacillus cereus). Upon examination of their 16S rRNA se−
quences and biochemical profiles, the six Antarctic bacterial isolates were identified as
Gram−negative Pedobacter cryoconitis (BG5), Pseudomonas migulae (WEK1), P. corru−
(WEA1) and Pseudomonas spp. (MTC3, MA2, and CG21). While inhibitors produced
by strains BG5, MTC3 and CG21 were sensitive to protease treatment, those produced by
strains WEK1, WEA1, and MA2 were insensitive to catalase, lipase, a−amylase, and prote−
ase enzymes. In addtion, the six Antarctic bacterial isolates appeared to be resistant to multi−
ple antibiotics.
Antarctica is the most pristine continent on Earth with a surface area of 14 mil− lion km2. The temperature in Antarctica is extremely low throughout the year ex− Pol. Polar Res. 32 (1): 3–14, 2011 Clemente Michael Vui Ling Wong et al. cept during the summer months where the ice on the soils is subjected to thawing(Convey et al. 2008). Due to the harsh conditions, microorganisms living in thecontinent and the neighboring islands have acquired unique adaptation strategiesto survive in the extreme environment. In order to gain competitive advantagesome microorganisms produce extracellular antimicrobial compounds to inhibitthe growth of their competitors (Russell 2006; Lo Giudice et al. 2007a). Theseantimicrobial compounds may have medical applications.
Over the last few decades microorganisms from around the world are being har− nessed for antimicrobial compounds. However, the antimicrobial activities of mi−croorganisms from the Antarctic, especially the bacteria, have only been investi−gated recently. Terrestrial (Moncheva et al. 2002; Nediakova and Naidenova 2004;O’Brien et al. 2004; Biondi et al. 2008) and marine bacteria (De Souza et al. 2006;Lo Giudice et al. 2007a, b) from several locations in the Antarctic are found to pro−duce a variety of antimicrobial compounds such as antibiotics and bacteriocins(O’Brien et al. 2004; Biondi et al. 2008). These reports indicated that there are sub−stantial numbers of novel bacteria with antimicrobial activities in the Antarctic. Thisopens up hope for the discovery of new classes of antibiotices considering that thereare only few that are found elsewhere over the past decades (Cassell and Mekalanos2001). Nevertheless, the geographical locations covered in Antarctic are limited. Forexample, there is little information pertaining to antimicrobial compounds of bacte−ria from other locations in Antarctica such as the South Shetland Islands. Hence, thisproject was conducted (i) to estimate the population of cultivable bacteria withantimicrobial activity from the soil samples collected from King George Island, Ant−arctic, and (ii) to characterize the bacteria with antimicrobial activity and theantimicrobial compounds they produce.
Isolation of Antarctic bacteria. — Soil samples were collected from King
George Island (62°09’30.0” S, 58°56’15.2” W), one of the South Shetland Islandsduring the 43rd Scientific Antarctic Expedition organized by the Instituto AntárticoChileno (INACH) in 2007. Samples were collected using sterilized spatula andstored in sterilized containers at −20°C for 10 to 14 days. Isolation of the Antarcticbacteria was performed using several growth media namely, Tryptic Soy agar(TSA) (Difco), Luria−Bertani agar (LBA), Nutrient agar and R2A agar (Difco).
One gram of the soil sample was inoculated into 10 ml of sterile distilled water orpotassium phosphate buffer (pH 7.2), serially diluted to 100 times and plated onthe agar medium. The agar plates were incubated at 20°C between two to ten daysfor the recovery of the Antarctic bacteria.
Detection of antimicrobial activity. — Antimicrobial compound production
was determined using deferred antagonism procedure (Kekessy and Piguet 1970).
Pseudomonas and Pedobacter isolates from King George Island Antarctic bacteria colonies on the agar plate were overlaid with 10 ml molten nutrientagar (1.3% nutrient broth and 0.7% agar) containing one of the indicator bacteria.
Zones of clearing around the Antarctic bacteria after 2 days of incubation indicatedthe presence of inhibitors. Indicator bacteria used were foodborne pathogens namely:Escherichia coli 0157:H7, E. coli V517, E. coli 0125, Salmonella enterica serovarTyphimurium (S. Tm 13), S. enterica serovar Typhi (S. Ty 10), S. biafra (S. Bi 8), S.
(S. Br. D), Klebsiella pneumoniae 14x, Enterobacter cloacae 22x,Vibrio parahaemolyticus 1808, V. parahaemolyticus 1896, V. parahaemolyticus2053, V. parahaemolyticus 2341 and Bacillus cereus K3. These foodborne pathogenswere provided by Professor Son Radu, Universiti Putra Malaysia, Malaysia.
Sensitivity of the antimicrobial agents to enzymes. — A series of enzymes
namely: protease (EC (Sigma), catalase (EC (Sigma), lipase(EC (Sigma), a−amylase (EC (Sigma), were used to determine theproperties of the antimicrobial agents produced by the Antarctic bacteria. All theenzymes were prepared at a concentration of 25 mg ml−1, according to the manu−facturer’s instructions. Deferred antagonism assays were performed according toO’Brien et al. (2004). The assay plate was incubated at 20°C for 12 hours.
Identification of the Antarctic bacteria. — Genomic DNA was extracted using
the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instruc−tion. PCR amplification of 16S rDNA was performed using the primers, BSF8/205’−aga−gtt−tga−tcc−tgg−ctc−ag−3’ and BSR1541/20 5’−aag−gag−gtg−atc−cag−ccg−ca−3’(Wilmotte et al. 1993). The PCR conditions were 96°C for 2 min, followed by 30 cy−cles at 96°C for 30 sec, 52°C for 40 sec, 72°C for 40 sec and a final extension step at72°C for 10 min. The 16S rDNA was sequenced using the Automated BiosystemsSequencer AB3100, and the sequence was analyzed using the BLASTn software(Altschul et al. 1997).
Biochemical profiles of the Antarctic bacteria. — Catalase activity was
tested according to the methods described by Lee et al. (2000), while the presenceof oxidase activity was tested according to the manufacturer’s instructions (bio−Merieux, Marcy−l'Etoile, France). Biochemical profiles of the bacteria were deter−mined using the API 20NE strips (bioMerieux). The incubation times of the APIstrips were between 4–5 days at 20°C. Motility test was conducted according to themethods described by Tittsler and Sandholzer (1936).
Antibiotic susceptibility and resistance tests. — Antibiotics susceptibility
test for the Antarctic bacterial isolates were performed by the disc diffusionmethod on tryptic soy agar medium. The antimicrobial agent tested includedampicillin (10 μg), ceftazidime (30 μg), imipenem (10 μg), ciprofloxacin (5 μg),chloramphenicol (30 μg), gentamicin (10 μg), kanamycin sulfate (30 μg), strepto−mycin (10 μg), tetracycline hydrochloride (30 μg), polymyxin B sulfate (300units), erythromycin (15 μg), clindamycin (2 μg), lincomycin (10 μg), novobiocin(5 μg), and vancomycin (30 μg).
Clemente Michael Vui Ling Wong et al. Host spectrum of inhibitor−producing Antarctic bacteria. — A total of
2465 Antarctic bacterial isolates were picked and screened for antimicrobial activ−ity against a series of foodborne pathogens. Six isolates, BG5, MTC3, WEK1,WEA1, MA2 and CG21 were found to inhibit four or more pathogen strains out ofthe 14 tested (Table 1). Isolates BG5 and MTC 3 inhibited the growth of 8 and 6strains of pathogens respectively. Isolates WEK1 and WEA1 inhibited the growthof 5 strains of pathogens while isolates MA2 and CG21 inhibited the growth of 4strains of pathogens.
Antarctic bacterial isolates WEK1, WEA1 and MA2 inhibited the growth of Gram−negative pathogens V. parahaemolyticus (1808, 2053 and 2341) and Gram−positive pathogen B. cereus (Table 1), but did not inhibit the growth of E. coli, Sal−monella spp., K. pneumoniae or E. cloacae. In contrast, isolates BG5, MTC3 andCG21 inhibited the growth of E. coli (O157:H7, V517 and 0125) and E. cloacae butnot V. parahaemolyticus (1808, 1896, 2053 and 2341) (Table 1). Bacterial isolateBG5 inhibited the growth of S. enterica serovar Typhimurium, S. enterica serovarTyphi, K. pneumoniae and B. cereus, while isolate MTC3 inhibited the growth of S.
serovar Typhimurium and K. pneumoniae but not B. cereus. None of theantimicrobial compounds produced by the six Antarctic bacterial isolates inhibitedthe growth of pathogens S. biafra and S. braenderup.
Partial characterization of the antimicrobial compounds. — The proper−
ties of the antimicrobial compounds of the 6 bacterial isolates BG5, MTC3,WEK1, WEA1, MA2 and CG21 were partially resolved by testing the sensitivitiesof these compounds towards several enzymes (data not shown). None of the Identification of antimicrobial producers against the foodborne pathogens, E. coli O157:H7, E. coli V517, E. coli 0125, S. enterica serovar Typhimurium (S. Tm 13), S. entericaserovar Typhi (S. Ty 10), S. biafra (S. Bi 8), S. braenderup (S. Br D), K. pneumoniae 14x,E. cloacae 22x, V. parahaemolyticus 1808, V. parahaemolyticus 1896, V. parahaemo−lyticus 2053, V. parahaemolyticus 2341 and B. cereus K3. Size of the inhibition zone of the foodborne pathogen (mm); (–), no inhibition zone pneu− cloa− V. parahaemolyticus isolates O157:H7 V517 0125 S. Tm 13 S. Ty 10 S. Bi 8 S. Br D 14X 22X 1808 1896 2053 2341 K3 Pseudomonas and Pedobacter isolates from King George Island antimicrobial compounds produced by the six bacterial isolates were sensitive totreatment with catalase, lipase, or a−amylase. However, the inhibitors produced bythe isolates BG5, MTC3 and CG21 were sensitive to protease while those pro−duced by the isolates WEK1, WEA1 and MA2 were not. Interestingly, the inhibi−tors of the six Antarctic bacterial isolates against the pathogens were affected at el−evated temperature. Inhibitory effect against the pathogens was detected at 20°Cbut not when the Antarctic bacteria were incubated at 30°C.
Identification of the Antarctic bacteria. — The 16S rDNA sequence (approxi−
mately 1.5 kb) alignment analysis of the six Antarctic bacterial isolates revealed thatfive isolates MTC3, WEK1, WEA1, MA2 and CG21 had high similarity to the gen−era: Pseudomonas from the phylum of g−Proteobacteria (Table 2). Bacterial isolatesMTC3, WEA1 and WEK1 had the highest similarity to the Pseudomonas sp.
DhA−91 (99%), Pseudomonas corrugata (99%) and Pseudomonas migulae (98%)respectively. MA2 and CG21 had the highest similarity to the Pseudomonas sp.
DM2 with 97% and 98% similarity respectively. The other isolate, BG5 was similarto the Pedobacter cryoconitis (99%) from the phylum of Bacteroidetes (Table 2).
The 16S rDNA sequences of the six bacterial isolates were deposited in theGenBank and assigned to accession numbers EU637886, EU637887, EU547450,EU547451, EU908689 and EU908688 for isolates BG5, MTC3, WEA1, WEK1,MA2 and CG21 respectively.
The 16S rDNA sequence affiliation of the six Antarctic bacterial isolates to their nearest Some of the 2465 Antarctic bacterial isolates, that had pigmented colony but did not exhibit any inhibitory effect against the foodborne pathogens tested, werealso identified. Although, these isolates did not inhibit the test pathogens used inthis study they might have an effect on other pathogens, because antibiotic pro−duction was linked to pigmentation of the bacterial strains (Choi et al. 2001; LoGiudice et al. 2007a). They were from the genera: Arthrobacter (10 isolates),Aeromicrobium (2 isolates), Brevundimonas (2 isolates), Cryobacterium (4 iso−lates), Dyadobacter (2 isolates), Flavobacterium (2 isolates), Methylibium (1isolate), Rhodococcus (7 isolates) and Sphingomonas (2 isolates) (16S rDNA se−quence data not shown).
Clemente Michael Vui Ling Wong et al. Characterization of the Antarctic bacteria. — All the 6 isolates BG5,
MTC3, WEK1, WEA1, MA2 and CG21 were Gram−negative bacteria. They didnot produce hydrogen sulfide or indole (Table 3); nor did they reduce nitrate, fer−ment glucose, assimilate adipic acid, trisodium citrate or phenylacetic acid. Theywere divided into two groups based on their characteristics.
Group one consisted of Pseudomonas spp. WEK1, WEA1 and MA2. They were motile, Gram−negative and grew on MacConkey agar medium. However, Morphologies and biochemical profiles of inhibitor−producing bacterial isolates: +, posi− Pseudomonas and Pedobacter isolates from King George Island they differed slightly in their morphologies and biochemical profiles. For exam−ple, isolate WEA1 was able to assimilate malic acid but not capric acid, and gavenegative reaction to esculin (Table 3). In contrast, isolate WEK1 was able to as−similate capric acid but not malic acid, and gave positive reaction to esculin. Un−like WEA1 or WEK1, isolate MA1 was able to assimilate capric and malic acids,and gave positive reaction to esculin. Additionally, isolates WEK1 and WEA1were spiral shaped while MA2 was rod shaped.
Pseudomonas spp. MTC3 and CG21, and Pedobacter sp. BG5 were assigned to another group. They were non−motile and did not grow on MacConkey agarmedium. The three isolates had similar biochemical profile, except that isolatesMTC3 and CG21 assimilated L−arabinose while the isolate BG5 did not (Table3). Additionally, MTC3 and BG5 assimilated N−acetyl−glucosamine but CG21did not. Isolates MTC3, CG21 and BG5 had rod, diplococcus and spiral shapesrespectively.
Antibiotics susceptibility. — The results of the antibiotic susceptibility test
for the six bacterial isolates BG5, MTC3, WEK1, WEA1, MA2 and CG21 areshown in Table 4. Interestingly, all the six isolates were resistant to ampicillin andvancomycin but were susceptible to imipenem, ciprofloxacin and tetracycline. Iso−lates WEK1, WEA1, MA2 and CG21 were resistant to 7 out of the 15 antibioticstested while isolates BG5 and MTC3 were resistant to 9 out of the 15 antibioticstested.
Antibiotics resistance profiles of the six bacterial isolates WEA1, WEK1, BG5, MTC3, CG21 and MA2. R, resistant; S, susceptible Clemente Michael Vui Ling Wong et al. Six out of 2465 isolates inhibited the growth of one or more foodborne patho− gens. Hence, the frequency of isolation of inhibitor producers in this study was 0.24% (6/2465 isolates) which was comparable to bacteria with antimicrobial com−pounds reported by Lo Giudice et al. (2007b) of 0.29% (13/4496 colonies) fromAntarctic marine samples. However, the numbers were lower than those reported byO’Brien et al. (2004) and Lo Giudice et al. (2007a) of 3.8% (22/580 colonies) fromthe east Antarctic terrestrial samples. In general, these observations suggest that thefrequency of isolation of inhibitor producers from Antarctic environment usingnon−selective media ranged from 0.2% (this work) to 3.8% (O’Brien et al. 2004; LoGiudice et al. 2007a).
The antimicrobial compounds from bacterial isolates MTC3 and BG5 had the broadest spectrum among the six isolates, inhibiting the growth of pathogens offour genera (Escherichia, Salmonella, Klebsiella and Enterobacter) and five gen−era (Escherichia, Salmonella, Klebsiella, Enterobacter and Bacillus) respectively(Table 1). On the other hand, antimicrobial compounds of bacterial isolatesWEK1, WEA1, MA2 and CG21 had narrow spectrum. They inhibited the growthof pathogens of two genera only.
All the six Antarctic isolates inhibited the growth of Gram−negative bacteria including pathogens from the genera Vibrio, Klebsiella and Enterobacter that hadnever been tested on Antarctic bacteria before. In contrast, most of the Antarcticbacteria reported by O’Brien et al. (2004) inhibited only Gram−positive bacteria.
Additionally, four isolates BG5, WEK1, WEA1 and MA2 also inhibited thegrowth of the Gram−positive foodborne pathogens and these findings were consis−tent with those reported by Lo Giudice et al. (2007b) although some of the indica−tor strains used were different.
The results from the enzyme sensitivity tests indicated that the active moieties of the antimicrobial compounds from the six isolates were not sensitive to catalase,lipase and a−amylase and therefore did not contain any hydrogen peroxide, lipid,or glycan. The inhibitors of the bacterial isolates BG5, MTC3 and CG21 were sen−sitive to protease and elevated temperature, suggesting that their inhibitors mightbe proteinaceous in nature and could be bacteriocins which were active only at anoptimal temperature (Leroy and De Vuyst 1999; Keren et al. 2004). Inhibitors ofthe isolates WEK1, WEA1 and MA2 were not affected by protease treatment andtherefore were not likely to be bacteriocins, but they were inactivated at elevatedtemperature. The reasons for the inactivation were unclear, and would only beknown when the inhibitory compounds from those bacterial isolates were purifiedand characterized.
The six Antarctic bacterial isolates were identified based on the alignments of their 16S rDNA sequences to those of known bacteria in the genebank to find theirnearest taxonomic neighbor (Table 2). Isolates WEA1, WEK1 and BG5 were Pseudomonas and Pedobacter isolates from King George Island likely to be P. corrugata, P. migulae and Pedobacter cryoconitis, respectively.
The other isolates MTC3, MA2 and CG21 were closest to the Pseudomonas spp.
and could be new pseudomonad species. These three isolates were likely to be dif−ferent species because of their different morphology, chemical and pathogen in−hibitory profiles. Isolates MTC3 and MA2 were rods while CG21 was a diplo−coccus. Isolate MA2 was motile, grew on MacConkey agar medium, and inhibitedthe growth of V. parahaemolyticus and B. cereus. In contrast, isolates MTC3 andCG21 were non−motile, did not grow on MacConkey agar medium and inhibitedthe growth of E. coli and E. cloacae. However, isolate MTC3 was different fromCG21 because of its ability to assimilate N−acetyl−glucosamine and inhibited anadditional pathogen, the S. enterica serovar Typhimurium.
The five Pseudomonas spp., MTC3, WEK1, WEA1, MA2 and CG21 were from the phylum g−Proteobacteria which was well known to produce bioactivecompounds. Sutherland et al. (1985) and Grgurina et al. (2005) had isolatedmupirocin and syringopeptin from g−Proteobacteria that showed inhibitory activ−ity against pathogens such as Haemophilus influenzae, Neisseria gonorrhoeae,Mycobacterium smegmatis and Staphylococcus aureus. In contrast, Pedobactersp. including isolate BG5 was among the few members from the Bacteroidetesphylum that produced antimicrobial compounds. To our knowledge, this is thefirst time that Pedobacter sp. is reported to produce inhibitors against humanpathogens.
The six isolates BG5, MTC3, WEK1, WEA1, MA2 and CG21 were found to be resistant to a series of antibiotics, a phenomenon which was also observed by Koboriet al. (1984) and Lo Giudice et al. (2007a). The ability of the six Antarctic bacterialisolates in this study to resist to ampicillin suggested that they probably produce b−lactamase to degrade the ampicillin in the agar medium. Their resistance to other b−lactam antibiotics, such as ceftazidime (extended cephalosporins), indicated thatthe Antarctic bacteria probably had broad spectrum b−lactamases. Nevertheless, ad−ditional test on other b−lactams is required to confirm this. The six bacterial isolatesin this study were resistant to at least 7 types of common antibiotics. Similar trendswere reported by Siebert et al. (1996). They found that some of the bacteria fromAntarctic sandstone in McMurdo Valley were resistant to one or more antibioticssuch as streptomycin, chloramphenicol, erythromycin, ampicillin, cycloserine andtetracycline. Multiple antibiotic resistant strains of environmental bacteria were alsofound in (i) the river and bay of Tillamork, Oregon (Kelch and Lee 1978), (ii) the icecore from the Greenland (Miteva et al. 2004), and the Arctic permafrost subsoil inSiberia (Mindlin et al. 2008). These observations implied that the environmentalbacteria were probably natural reservoirs of antibiotic resistant genes.
In this study, six or 0.24% of the 2465 bacterial isolates were found to produce antimicrobial compounds against 13 out of the 14 indicator foodborne pathogens.
Five of the isolates were from Pseudomonas spp. and one from Pedobacter sp. Al−though bacterial isolates from other genera such as Arthrobacter, Aeromicrobium, Clemente Michael Vui Ling Wong et al. Brevundimonas, Cryobacterium, Dyadobacter, Flavobacterium, Methylibium, Rho−dococcus and Sphingomonas were also found in the same soil samples. They did notproduce any antimicrobial compounds against any of the 14 indicator pathogens butthey were pigmented, and pigmented bacteria had been linked to the production ofantibiotics (Shivaji et al. 1989; Shivaji et al. 1994; Chattopadhyay et al. 1997; Choiet al. 2001; O’Brien et al. 2004; Lo Giudice et al. 2007a). Probably these isolatesproduced antimicrobial compounds with narrow spectrum that targeted pathogensother than those used in this study.
The results from this study showed that the Antarctic bacteria produced antimicrobial compounds, either of broad or narrow spectrum, that targeted a widerange of pathogens. Apart from that, the six Antarctic bacterial isolates were resis−tant to multiple antibiotics suggesting that these Antarctic bacteria are potentialsources of genes encoding for both antimicrobial compounds and resistance to an−tibiotics for medical applications. From the ecological point of view these two ca−pabilities probably provided the competitive edge to the Antarctic bacteria to sur−vive in the harsh environment (Russell 2006; Lo Giudice et al. 2007a), while bac−teria lacking such capabilities would be suppressed.
Acknowledgements. — This work was funded by the Malaysian Antarctic Research
Programme (MARP), Ministry of Science, Technology and Innovation (MOSTI), Malaysia andInstituto Antártico Chileno (INACH), Chile. The authors would like to thank personnel atINACH especially José Retamales, Marcelo Leppe, Paulina Julio Rocamora, Verónica Vallejos,Cristian Rodrigo and Patricio Barraza for advice and logistic support.
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