Inter-genic Cross-Infectivity of Industrial Wastewater Bacteriophage between Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 and Pseudomonas aeruginosa PAO303 JS Sunico and RG Gicana ABSTRACT
Most bacteriophages are host-specific. However, there exist reports on the prevalence of wide host-range lytic bacteriophages infecting Pseudomonas and E. coli (Jensen et al., 1998). This research confirmed reports on the inter-specific and inter-genic cross-infectivity of a wild-type bacteriophage isolated from an industrial sewage to transfer genes by lysogenic conversion from Enterohemorrhagic E. coli (EHEC 0157:H7) and Pseudomonas aeruginosa PAO303. Transduction of EHEC gene into Pseudomonas pose a very important medical issue since this organism is among the most widespread nosocomial infections. To detect the transductants, antibiotic markers were used: Rifampicin-resistance (Rifr) for Escherichia coli and Streptomycin-resistance (Strr ) for Pseudomonas aeruginosa. Of the 1,056 Pseudomonas isolates screened, 12 colonies were considered putative transductants. Of these, two (2) were antibioticgene transductants with characteristic StrsRifr. The 10 other isolates were StrsRifs indicating that they were transductants other than of the antibiotic-resistance genes. Biochemical confirmation of these transductants proved that they were Pseudomonas aeruginosa.
INTRODUCTION Bacteriophages are obligate intra-cellular parasites of bacteria. They are among the most important agents for the evolution of new traits among medically-important organisms. Some of the bacteriophage-mediated pathogenicity are the β-bacteriophage of Corynebacterium diphtheriae, shiga toxin of Shigella dysenteriae and the verotoxin of Enterohemmorhagic E. coli. Interaction between the bacteriophage and host receptor is almost always very specific. Thus, bacteriophage infection is almost always limited to the host with a specific surface receptor; otherwise there is no host infection. Humans and warm-blooded animals carry Escherichia coli in the intestinal tract as harmless commensals. Some strains, however, such as Enterohemorrhagic E. coli (EHEC) can cause severe food-borne disease. EHEC produces toxins, known as verotoxins or shiga-like toxins because of their similarity to the toxins produced by Shigella dysenteriae (Duyen, 2007). EHEC 0157:H7 is the most important serotype in relation to public health and is easily differentiated biochemically from other E. coli strains. The reservoir of this pathogen appears to be mainly cattle and other ruminants. It is transmitted to humans
primarily through consumption of contaminated foods such as raw or undercooked ground meat products and raw milk. Fecal contamination of water and other foods, as well as cross-contamination during food preparation also leads to infection. Examples of foods implicated in outbreaks of E.coli 0157:H7 include undercooked hamburgers, yogurt, cheese and milk. EHEC has also been isolated from bodies of water, wells, and water troughs, and had been found to survive for months in manure and water-trough sediments (Muniesa et al., 2006). On the other hand, Pseudomonas aeruginosa is a bacterium that does not possess EHEC gene but is most commonly isolated in patients with surgical wound infections, ventilator-associated pneumonia and urinary tract infections. Although nosocomial (hospital-acquired), this is also common putrefying organism in refrigerated meat products responsible for the spoilage of foods. Given its psychrophilic nature, Pseudomonas aeruginosa can cause food poisoning upon ingestion of improperly prepared foods. There has been no report as yet of the transfer of EHEC gene to other bacterial species to occur in nature and so, this research presupposes the potential harm of the transfer of EHEC gene to Pseudomonas can do to humans. The transfer of EHEC genes by transduction to occur in nature poses a great alarm to researchers that some genes carried by E. coli infected by a bacteriophage can be transferred to other species which are normal flora of the human body. It is to this premise that this research was conducted. Objectives: This study aimed to determine the inter-genic cross infectivity of a wild-type bacteriophage isolated from industrial sewage between Enterohemorrhagic Escherichia coli (EHEC) and Pseudomonas aeruginosa. This study specifically aimed to: 1. isolate a wild-type bacteriophage from industrial sewage; 2. test the bacteriophage for inter-genic host infection; 3. randomly transduce EHEC genes into Pseudomonas aeruginosa 4. screen for transductants
MATERIALS AND METHODS Bacteriophage Isolation Bacteriophages were isolated by an enrichment procedure described by Jensen, et al. (1998). Sewage water samples collected from the Sewage Treatment Plant of Asian Alcohol Corporation in Pulupandan, Negros Occidental was amended with an equal volume of growth medium Luria broth. Enrichment cultures were incubated overnight at 37°C with shaking. After incubation period, samples were treated with 1/20 volume of chloroform and centrifuged to eliminate all viable bacterial cells. The bacteriophage isolate was purified. Bacteriophage Purification
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Bacteriophage lysates were produced by a modification of the just-confluent-lysis protocol of Aber et al. (1983). Appropriately diluted samples containing bacteriophages were mixed with 0.1 ml of overnight bacterial host culture and plated with 3 ml top agar. Bacteriophage preparations were mixed and quantified by plating appropriately diluted samples of bacterial cells. Bacteriophages were purified by performing a minimum of three rounds single-plaque isolation. Host Specificity Assay Bacteriophages were checked for its host-specificity by spot lysis analysis on EHEC O157:H7 and Pseudomonas aeruginosa (PAO303) (Adapted from Jensen, et al., 1998). Spot lysis test done on Escherichia coli and P. aeruginosa were formed by plating samples of overnight cultures mixed with top agar onto appropriate agar plates. After the top agar solidified, samples of bacteriophage lysates were dropped onto the agar and the plates were incubated overnight. Productive interaction of bacteriophage with the host bacterium was revealed a zone of plaque formation at the site of virus addition (Jensen et al., 1988). PFU Determination of Bacteriophage Inoculum Counting of the Plaque Forming Units (PFU) was done to make sure that the phages were effectively causing infection. The number of phage necessary for transduction to occur was about 1 X 106 PFU/ml.
PFU / ml
Where: PFU/ml P df V
( P)(df ) V
= Plaque forming unit per milliliter = Average number of plaques = dilution factor = Volume of sample plated
Serial dilution was performed to ensure that the phage count falls within the range of 1 x 105 to 1 x 106 PFU/ml. Plaque Assay Well-isolated plaques were cut from agar plates, placed in a sterile diluent (MC buffer), and used to produce new bacteriophage lysates. These procedures were repeated through three cycles until lysates produce single plaque morphology. Preparation of Phage Lysate To prepare a phage lysate, a 0.1 ml of an overnight broth culture of Rifr-EHEC was mixed with 0.1 ml 106 PFU of the bacteriophage. This was incubated for 20 min at 37°C water bath to allow adsorption of phage to bacterial cells. During the course of
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incubation, random encapsulation occurred between the phage and E. coli. This is called generalized transduction. As soon as incubation was completed, a 5 ml of molten soft agar was added and immediately poured on a fresh R-base agar plate. The transduced cells were collected by adding 5 ml sterile MC buffer onto the top agar, scraped, added with 1.0 ml chloroform, mixed and centrifuged at 4,000 rpm for 15 minutes. The supernate that supposedly contained the bacteriophage was collected and purified by adding 1 drop of chloroform The number of phage particles per ml was determined by serially diluting the stock using MC buffer at dilutions of 10-2, 10-4, 10-6, and 10-7 and was mixed with 0.1 ml of each serial dilution with an overnight culture of Rifr-EHEC. The plaque forming units (PFU) was determined using an agar-overlay method. In this method, 3 ml of top agar was added and spread on base agar. The agar plates were incubated overnight to allow plaques to develop. The sterility of the lysate was tested by planting in 0.1 ml on Luria agar. Transduction To perform transduction, 2 ml of an overnight culture of Streptomycin resistant Pseudomonas aeruginosa (Strr-PAO 303) was added to 2 ml of previously prepared RifrEHEC phage lysate. The incubated PAO303 was incubated at 37°C water bath for 20 min to allow the adsorption of phage to bacteria. This was then centrifuged for 15 min at 8,000rpm. Supernatant was discarded and the pellet was resuspended in 2 ml saline. A series of dilution was made on the suspension 10-5, 10-6 and 10-7 dilutions were plated on Luria agar in triplicates. The plates were incubated overnight for single isolated colonies to grow. Colonies were picked and screened for possible transductants. Screening by Lederberg and Lederberg Replica Plating Technique Selection and identification of different phenotypes of organisms is important for microbiologists in the field of research. Lederberg and Lederberg Replica Plating Technique is very useful in such determination of the physiological traits. For the replica plating technique, a replicator consisting of sterile velveteen material attached to a prong inoculator is commonly used. For this research, a multiprong stainless steel inoculator was used. This method proved useful in the selection of transductants (Raymundo, 1991). The replica plating technique permits the simultaneous transfer of a large number of colonies from one plating medium to another in a single operation. Furthermore, the exact pattern of colonies on the master plate is reproduced on the replica plate, thus permitting the rapid examination of large numbers of individual colonies for mutant characteristics. A master plate contains a complete medium (e.g. Luria agar) and is made by inoculating individual colonies from the unknown mutants or samples with the use of sterile toothpick. To ensure that colonies are inoculated corresponding to the individual tooth of the prong, a grid is especially made. During the incubation period, the proper incubation time was followed to prevent over-growths and the spreading of the colonies. The transferring of the colonies with the use of the prong inoculator from the master plate to the different media was done in order of increasing antibiotic susceptibility.
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Inoculation started from the agar medium containing antibiotics where PAO303 was resistant (LAstr) to the agar medium where PAO303 was susceptible (LArif). Colonies that grow to certain medium only are those strains which are resistant to certain antibiotics, chemicals or other antibacterial agents, or those that are capable of utilizing that medium (Raymundo, 1988). Screening of transductants was performed using the Lederberg and Lederberg replica plating technique whereby a 2.0 ml aliquot from a suspension of transduced Pseudomonas aeruginosa PAO303 was diluted in saline solution and was plated onto LA + Streptomycin (LAS), LA + Rifampicin (LAR), and LA + Streptomycin + Rifampicin (LARS) all in triplicates. The same technique was done with 0.1 ml of phage suspension to confirm sterility and 0.1 ml of Streptomycin resistant Pseudomonas aeruginosa (StrrPAO 303) for spontaneous reversion. Twenty-four (24) master plates using Luria agar were prepared containing 1056 potentially transduced colonies. Using sterile toothpicks to transfer single colonies onto LA agar with grids, the isolates were assumed to be pure or axenic culture. Assuming that one colony arises from one single cell, then each colony is of unique morphology. After 24 hours of incubation on the master plates, each of the 528 isolates on 24 master plates were replica plated onto LA + Str, LA + Rif, LA + str + Rif and LA alone. This research was conducted at the Medical Technology laboratory of Riverside College, Bacolod City.
RESULTS AND DISCUSSION Using the enrichment technique for the isolation of bacteriophage from industrial wastewater, results of seven trials produced plaques with count of 5.28 x 1011 PFU/ mL.
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The plaques were small (pinhead sized) with smooth plaque edges. The host used for the determination of the plaques was the EHEC. To test the bacteriophage for inter-genic host infection, the isolated and purified bacteriophage was used to infect two different hosts. Figure 1 showed the plaque formation both for EHEC and Pseudomonas aeruginosa.
Figure 1. (a) Plaque formation (arrows) on EHEC 0157:H7 and (b) Pseudomonas aeruginosa PAO303
Upon confirmation of the inter-genic host infectivity of the isolated bacteriophage, transduction was performed. From the twenty four master plates prepared containing 44 colonies each, 36 potential transductant colonies were isolated. Of these two were rifampicin gene transductants (Figure 2).
Figure 2. Possible transductants (arrow) on LAR, LAS, LARS and Luria agar using Lederberg and Lederberg Replica Plating Techniques
Isolation of EHEC genes from a pool of many fragments of gene in E. coli is a probability event. Using bacteriophage with probably a high generalized transducing ability, the transductants were isolated, screened and confirmed to be Pseudomonas aeruginosa which acquire a new trait, rifampicin resistance and streptomycin susceptibility. The phenotypic descriptions of each putative transductant for Rifampicin gene are shown in Table 1. Table 1. Characterization of PAO303 transduced with Rifr gene from E. coli.
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Code JsP-4 JsP-9
Phenotype Str-Rif+ Str-Rif+
Interpretation Transduction of the rif gene Transduction of the rif gene
The two isolates described above result from the random packaging of bacteriophage of genes coming from Escherichia coli. This result supplements the contention that transduction actually occurred. Isolation of Str-resistance gene and insertion of this into Pseudomonas aeruginosa is an indication of random isolation of EHEC gene from Escherichia coli. General transducing phage packaging system is not very fastidious, so the size of the DNA molecule does not have to be exactly the same in all particles. The result is that in the population particles produced when infected bacteria lyse, there are generalized transducing particles, the major class of which contains a single fragment of bacterial DNA. Fragmentation of the host DNA is random process and the transducing particles contain fragments derived from all regions of the host. Thus, a sufficiently large population of P1-progeny will contain at least one particle possessing each host gene. On the average, for any particular gene, there is roughly one transducing particle per 10 6 viable phage. Generalized transducing particles do not produce the bacteriophage progeny because they contain no bacteriophage DNA; however, the bacterial DNA is injected into the host cell (Freifelder, 1987). Resistance to antibiotics is usually carried in the genes of bacteria. Escherichia coli were detected to be resistant to rifampicin by their growth on Luria agar + rifampicin medium. On the other hand, Pseudomonas was detected to be streptomycin resistant by the growth on Streptomycin antibiotics. To address the third objective, Table 2 showed the different transductants probably harboring EHEC genes. Table 2. Characterization of PAO303 transduced randomly with various gene fragments from E. coli Code JsR-1 to JsR-10 Phenotype Str-RifInterpretation Str gene damaged; Rif gene was not transduced; Insertion of other gene fragments
If the integration site for any gene introduced into Pseudomonas resides at the gene maps linked to antibiotic resistance for streptomycin, then insertion into this site will render the bacteria susceptible to streptomycin when grown on streptomycin agar.
Thus, Figure 3 illustrates transductants for any genes other that rifampicin resistance gene probably inserting the EHEC gene.
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a (b)
Figure 3. Possible transductants; (a) StrsRifs resulting from generalized transduction of genes other than Streptomycin-resistance; (b) StrsRifr resulting from transduction of Rifampicin gene into Pseudomonas aeruginosa These organisms when grown on LA + Streptomycin, LA + Rifampicin and LA + Streptomycin and Rifampicin will not grow because: (1) their genes for streptomycin resistance was damaged due to integration; (2) the inserted gene is not rifampicin resistance gene. Finally, to ensure that the isolated transductants were really Pseudomonas aeruginosa morphologic and physiologic tests were performed. Confirmatory tests included were Carbohydrate fermentation test using triple sugar iron agar (TSI), lactose fermentation using Mac Conkey Agar (MaC) and Oxidative Fermentative Medium (Hugh and Leifson Medium). Confirmatory tests were presented in table 4. Table 3. Confirmatory test results of the different transductants Code JsP-4 JsP-9 JsR-1 JsR-2 JsR-3 JsR-4 JsR-5 JsR-6 JsR-7 JsR-8 JsR-9 JsR-10 Phenotype Str-Rif+ Str-Rif+ Str-Rif-Str-RifStr-RifStr-RifStr-RifStr-RifStr-RifStr-RifStr-RifStr-RifTSI K/K K/K K/K K/K K/K K/K K/K K/K K/K K/K K/K K/K MaC NLF NLF NLF NLF NLF NLF NLF NLF NLF NLF NLF NLF O/F O O O O O O O O O O O O Remarks TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL TYPICAL
Based on the tests, all of the isolates were confirmed to be Pseudomonas and not E. coli nor other contaminants. Triple Sugar Iron Agar is a butt-slant medium containing three sugars, glucose (0.1 %), lactose (1 %) and sucrose (1%) with phenol red as the redox indicator. Glucose utilization by certain microorganisms like E. coli occurs both aerobically in the slant and
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anaerobically in the butt. Once glucose is fermented, acids will be produced. These acids in the medium will cause the phenol red to be reduced to yellow color. Thus, the butt slant will appear yellow after 6 hours of incubation. (Delost, 2004). Pseudomonas aeruginosa does not ferment any sugar aerobically, hence, there is no change in the color of the TSI. On the other hand, Hugh and Leifson Oxidative-Fermentative (OF) medium test the type of sugar metabolism of microorganism. It contains the redox dye Bromthymol blue which is green in its oxidized state but yellow when reduced. Acid production either fermentatively or oxidatively will change the color of OF medium from green to yellow. Pseudomonas aeruginosa does not produce acid either oxidatively or fermentatively, hence, OF(-/-). On the otherhand, E. coli reduces the BTB dye both oxidatively and fermentatively, hence, OF (+/+) Delost, 2004). Moreover, McConkey is a culture medium which differentiates lactose fermenters from non-lactose fermenters. It contains crystal violet and bile salts as inhibitory substance against gram-negative, non-lactose fermenters. Neutral red is the indicator dye used which turns the color of the colony into red in acid environment and yellow in nonacid environment. Hence, E. coli is a lactose fermenter giving pink colonies while Pseudomonas aeruginosa is non-lactose fermenter producing yellow colonies (Delost, 2004). Beyond the scope of the research, was an atypical change in the color of transductant colonies was observed. Increased pigment production of the transduced strains (Figure 4) was observed.
Figure 4. Increased pigment production in Pseudomonas aeruginosa JsR-1 compared to the wild type Pseudomonas aeruginosa PAO303
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS This experimental research generated the following findings or results of biological significance:
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(1) (2)
(3)
a general transducing bacteriophage was successfully isolated from industrial sewage; the bacteriophage was infective against two different genera of bacteria: E. coli and Pseudomonas aeruginosa showing its broad-range of hosts or inter-genic host infectivity; twelve transductants were isolated: two of which were for transduction of the rifampicin gene and ten were the transductants for genes other than rifgene inserted near or at the str-gene as the site of cleavage;
Based on the results of the screening, the researcher had successfully isolated, screened and partially characterized twelve (12) transductants but these transductants are still subject for further molecular study for the transfer of EHEC characteristic to Pseudomonas aeruginosa. Furthermore, this research is a confirmation of the previous studies published by Ellen C. Jensen, et al. in the American Society of Microbiology Journal dated February 1998 of the inter-specific cross infection between Escherichia coli and Pseudomonas aeruginosa. Recommendations In the light of findings and conclusions of this study, the researcher made the following recommendations: 1. That the bacteriophage be screened by electron microscopy and other serologic technique for its identity; 2. That the isolated transductants will be further screened by other researchers using molecular techniques for the transfer of EHEC characteristics to Pseudomonas aeruginosa; 3. A host infection assay or animal inoculation assay would be also recommended to other researchers for further study and confirmation; 4. That fast food stores will be cautious in proper food preparation and handling since the presence of these organisms might be one of the mechanisms of the emergence of the disease and strictly employ HACCP (Hazard Analysis and Critical Control Point) practices; 5. Based on recent discoveries in changes of the physiology of P. aeruginosa, research on change in membrane/cell wall composition be conducted to evaluate the reasons for increased pigment production and resistance to infection.
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