Detection of AmpC Beta-Lactamase genes in enteric siolates using multiplex PCR
Connell, Jennifer D.
Both communicable and nosocomial infections involving ampC Beta-lactamase producing bateria are becoming more prevalent in the United States. Bacterial ampC Beta-lactamase genes may be plasmid-mediated, which can enhance spreading of these genes between bacteria, thereby increasing the prevalence of infections that are difficult to treat. A key step in limiting the damage done by these bacteria is the development of efficient isolation and analysis techniques, as more knowledge of these bacteria may be acquired at a quicker rate. Multiplex PCR is one technique which, when completed correctly, can yield a lot of results at once. Six control E.coli strains, each containing a different beta-lactamase resistance gene, were obtained from Dr. Nancy Hanson (Creighton University), to optimize the multiplex PCRs. Bacteria from human samples were subjected to the multiplex PCR using primers of the six control genes to see if beta-lactamase genes were present.
DETECTION OF AMP‐C BETA‐LACTAMASE GENES IN ENTERIC ISOLATES USING MULTIPLEX PCR Jennifer D. Connell and John C. Mullican Department of Biology, Washburn University, Topeka, KS 66621 Abstract: Both communicable and nosocomial infecGons involving ampC Beta‐lactamase producing bacteria are becoming more prevalent in the United States. Bacterial ampC Beta‐lactamase genes may be plasmid‐mediated, which can enhance spreading of these genes between bacteria, thereby increasing the prevalence of infecGons that are dificult to treat. A key step in limiGng the damage done by these bacteria is the development of eficient isolaGon and analysis techniques, as more knowledge of these bacteria may be acquired at a quicker rate. MulGplex PCR is one technique which, when completed correctly, can yield many results at once. Six control E. coli strains, each containing a different beta‐lactamase resistance gene, were obtained from Dr. Nancy Hanson (Creighton University), to opGmize the mulGplex PCRs. Bacteria from human samples were subjected to the mulGplex PCR using primers of the six control genes to see if beta‐lactamase genes were present. Methods Obtaining Control DNA and primers Control E. coli were obtained from Dr. Nancy Hanson on blood agar plates; each strain containing a plasmid with one of six parGal resistance genes present. A porGon of the amp‐C beta‐lactamase gene MOX, FOX, DHA, CIT, ACC, or EBC is contained on a plasmid along with a Kan‐resistant gene. The KAN resistance was used to grow these bacteria on LB KAN plates, forcing the bacteria to keep the plasmid containing the resistance genes. IniHal Monoplex and MulHplex PCR The control DNAs were subject to a monoplex PCR reacGon using a colony cell lysate of each. Also, a mulGplex reacGon was performed in which DNA and primers from each were used in equal amounts, respecGvely. PCR products were electrophoresed through 0.8% PCR grade agarose gels. Control Colony Lysate Plasmid Preps In order to avoid using a colony lysate in every control reacGon and for PCR opGmizaGon purposes, plasmid DNA preps were performed on each of the six control E. coli strains. A 0.8% agarose gel was performed on the plasmid preps. For bands that were not clear, steps were taken to opGmize the preparaGons. Plasmid preps for each control DNA were then diluted into 10‐2 and 10‐3 soluGons and stored to be used as control DNAs in the mono‐ and mulGplex PCRs. Monoplex and MulHplex PCR with Control Plasmid DNA Both monoplex and mulGplex PCRs of both 10‐2 and 10‐3 diluGons of the control DNAs were performed, followed by electrophoresis through a 1.5% PCR grade gel. A high percent of agarose is used in order to opGmize spacing and clarity of the bands. Obtaining Isolates in Humans Human samples were obtained from volunteers and streaked for isolaGon onto BE (Bile Esculin) plates, which select for Gram posiGve Enterococcus sp. Once isolated, samples were placed on an LB grid as well as an LB AMP grid. MulHplex PCR ReacHons with Human Isolates A colony lysate procedure was performed on 33 human isolate samples derived from the BE plates. This lysate was used in a mulGplex PCR reacGon. A control mulGplex reacGon was also performed with 5 of the 6 control DNAs. All PCR products were run through a 2.25% PCR grade agarose gel. Figure 3 displays results of these reacGons. Results and Conclusions • The plasmid prep procedure, as shown in Figure 1, yielded excellent plasmid preparaGons for all clones, but ACC. Another plasmid prep was performed on the clone containing the recombinant ACC gene. • As seen in Figure 2, all bands are present in the mulGplex PCR except for MOX. However, MOX can be observed in the monoplex PCR reacGon (Figure 2, lane 3). In order to solve this problem, primers for MOX were doubled while all other amounts of primers remained the same. However, MOX was sGll not at all visible. Therefore, when the control isolates were subject to mulGplex PCR, the MOX gene was likely not adequately analyzed. • Figure 3 displays the results of the human isolates subject to mulGplex PCR, indicaGng that none of the five control genes were present in any of the isolates. • Many of the human isolates grew very well on the LB AMP grid (Figure 4), indicaGng resistance to ampicillin. Further ExperimentaHon None of the control resistance genes may have been found in the Gram‐posiGve Enterococci isolates because the control genes used are more prevalent in Gram‐negaGve enteric bacteria . Therefore, further experimentaGon will include using the above procedures to test for resistance in Gram‐negaGve human isolates, pending IRB approval, using EMB plates, which are commonly used to select for Gram negaGve bacteria, including members of the Enterobacteracea family. In order to opGmize the mulGplex PCR with the MOX gene present, several acGons may be taken. The clone containing the control MOX gene DNA may be re‐grown and another plasmid prep performed to ensure that the control DNA for MOX used in each reacGon is in the best possible condiGon. Also, variaGon of the mulGplex PCR may be performed using different amounts of primers to opGmize the procedure. Another step that may be taken is splihng up the mulGplex into two reacGons with three genes each, or three reacGons with two DNA each as to observe if MOX will work with all or some of the other genes. Ideally, a single mulGplex reacGon would be preferred and we will conGnue to opGmize the condiGons for such a reacGon. IntroducHon Beta‐lactam drugs are commonly used in the treatment of many bacterial infecGons and include penicillins, cephalosporins, monobactams, and carbapenems. Beta‐lactam drugs act by inhibiGng the pepGdoglycan layer formaGon in cell wall, therefore blocking the division of the bacterium. The mechanism of this acGon depends on the inhibiGon of penicillin binding proteins (PBPs). The beta‐lactam molecule is able to irreversibly bind to the acGve site of the PBP, and thus not allowing the final cross linking of the pepGdoglycan layer to form. The efecGveness of beta‐lactam drugs rests in their ability to reach the PBP and to bind to the PBP. Therefore, any bacterium that is able to inhibit one of these acGons is able to cause resistance to the anGbioGc. Some bacteria may have the ability to produce enzymes, known as beta‐lactamases, which hydrolyze the beta‐lactam ring of the anGbioGc and render it inefecGve. The genes which express the beta‐lactamase genes may be either chromosomal or plasmid‐mediated. Another mode of resistance may be a result of the alteraGon of PBPs, on which beta‐lactam anGbioGcs are not as efecGve. DetecGon of these beta‐lactamase genes in an organism is important, as therapy methods difer between them. MulGplex PCR is one technique that may be used to test for mulGple resistance genes in one reacGon. To perform a mulGplex reacGon, forward and reverse primers of each gene of interest must be present, as each work simultaneously on available DNA. A control must also be conducted in which DNA, containing all or a segment of the resistance gene, is present in the reacGon as well as the forward and reverse primers for each gene. Lane 1: 1 kbp DNA Ladder Lane 2: MOX Lane 3: CIT Lane 4: DHA Lane 5: ACC Lane 6: EBC Lane 7: FOX Lane 1: PCR DNA Marker Lane 2: NegaHve Control (No DNA) Lane 3: MOX Monoplex Lane 4: CIT Monoplex Lane 5: DHA Monoplex Lane 6: ACC Monoplex Lane 7: EBC Monoplex Lane 8: FOX Monoplex Lane 9: MulHplex with 10‐2 Lane 10: MulHplex with 10‐3 Lane 11: MulHplex Control (No DNA) Lane 1: PCR Grade Marker Lanes 2‐20: Human Isolates Lane 21: PCR Grade Marker Lanes 22‐35: Human Isolates Lane 36: MulHplex PosiHve Control Lane 37: MulHplex NegaHve Control Figure 4: Bacterial Colonies RepresenHng Human Isolates on LB (le]) and LB AMP (right) Acknowledgements. We wish to thank Dr. Nancy Hanson of Creighton University for supplying the control bacteria and Dr. Andrew Herbig for helpful discussions and assistance in obtaining the human samples. Reference 1. Perez‐Perez, F. J., and N. D. Hanson. (2002) DetecGon of Plasmid‐Mediated AmpC Beta Lactamase genes in Clinical Isolates by using MulGplex PCR. Journal of Clinical Microbiology 40 (6):2153‐2162. Figure 1: Plasmid DNA Analysis via 0.8% Agarose Gel Electrophoresis 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 Figure 2: Monoplex and MulHplex PCR on Control DNA on a 1.5% PCR grade Agarose Gel Figure 3. Colony MulHplex PCRs on Human Isolates (BE Plates) Electrophoresed through a 2.25% PCR Grade Agarose Gel 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 http://www.life.umd.edu/classroom/bsci424/Images/PathogenImages/BetaLactam.gif http://www.life.umd.edu/classroom/bsci424/Images/PathogenImages/Penicillin.gif GENETIC AND MORPHOLOGICAL IDENTIFICATION OF FREE-LIVING AMOEBOFLAGELLATES FROM FRESHWATER SOURCES IN KANSAS Allison McKinley, Trent Snyder and John Mullican Department of Biology, Washburn University, Topeka, KS Abstract.. N. fowleri is a ubiquitous free‐living thermotolerant amoeboflagellate know to be the causative agent of primary amoebic meningoencephalitis (PAM), a rare but almost fatal disease of the central nervous system. Diagnosed incidences of PAM have been reported in the border states of Missouri and Oklahoma, but not in Kansas. Detection of N. fowleri in Kansas would suggest that individuals are potentially at risk of acquiring PAM. To determine whether N. fowleri exists in Kansas, water samples from five different fresh water reservoirs were obtained. These samples yielded a total of 122 amoeba isolates, 83 of which were found to be thermotolerant to 42˚C. Testing for the presence of N. fowleri was accomplished through a variety of morphological characteristics and genetic testing. Seventy one percent (59/83) of the thermotolerant isolates were able to transform into flagellates. DNA samples of each isolate were then subjected to N. fowleri ‐specific PCR. Results In total, well over 122 plaque isolates were obtained from 15 water samples collected from seven different freshwater sources (Table 1). While analysis is ongoing, approximately 69% (83 of 120) of those analyzed to date were found to be able to grow at temperatures of 42˚C. Approximately 71% (59/83) of the thermotolerant isolates were able to transform into flagellates. These findings were not surprising since the water samples were obtained during the warmer summer months. Figure 2 depicts a single amoebae plaque (left panel) and magnified views of cysts and amoebae from a growth plate. Preliminary analysis of isolates using gel electrophoresis has provided us with quantitative and qualitative data of each DNA purification sample collected from the individual amoeba isolates. Figure 3 contains photographs of five agarose gels (A-E) used to evaluate the quality and quantity of DNA from each amoeba sample. The bands near the wells, traveling the slowest and therefore the shortest distances, are representative of the high molecular weight genomic DNA purified from the amoeba isolates. The banding located furthest down the lanes represents the presence of RNA and/or DNA fragments due to shearing. Materials and Methods Environmental Sampling Each water sample was collected in separate sterile 50 mL centrifuge tubes placed under the water surface within approximately 0.3 m from the edge of the water line. The temperature and pH of the water from which the samples were obtained were recorded at each collection, along with the time of day. The samples were collected from sites where human exposure was a strong possibility with the exception to Central Park Fishing Lake where there is currently a posted sign advising the public against swimming or wading. Locations of Sampling Sites Processing Samples The collected samples were vortexed to resuspend sediments and 1.0 mL aliquots were pipetted onto NM agar plates containing a lawn of UV-killed Enterobacter aerogenes. The plates were then incubated at 37°C for a minimum of 24 hours. Additional time was allowed if amoebae plaques were either not present or smaller than 1.0 cm in diameter. Plaque isolation Individual clearings of the bacterial lawn (plaques) were transferred to a second plate by cutting a cube of agar from the original plate with a sterile stainless steel spatula, and positioned with the amoeba-side down on a new agar plate covered with an inactivated bacterial lawn. The process was repeated until pure isolates, free from any contaminants, were obtained for each original plaque sample. The pure isolate plates were incubated at 37°C and allowed to grow until the outgrowth plaques were large enough to obtain an agar cube to inoculate another plate with the intention of assessing temperature tolerance. A scraping was also collected using a sterilized inoculation loop for the purpose of inoculating an enriched liquid media to determine flagella morphology and for the purpose of extracting DNA. Assessing Temperature Tolerance Each isolate from the previous step was transferred to two separate plates. One plate was incubated at 37°C and one at 42°C for at least 48 hours. Any plates containing plaques that continued to expand in 42°C after 48 hours (“thermotolerant samples”) were removed and stored or directly transferred to liquid culture medium. A master pure isolate sample was kept in the refrigerator for the duration for future work. Liquid Cultures Samples containing thermotolerant amoebae were transferred to a liquid nutrient medium in a four chambered glass slide using a sterilized inoculation loop. The liquid media consisted of 5.0 mL Nelson’s Growth Medium (4) supplemented with 2% calf serum. All samples were incubated at 37°C overnight. The media was removed and replaced with sterile water which would promote flagella morphology by restricting nutrient concentration. DNA Extraction & Polymerase Chain Reaction Total Naegleria DNA was extracted using MasterPureTM DNA Purification Kit and following the manufacturer’s protocol (EpiCentre™ Technologies). DNA quantification and quality assurance was performed by running a small sample of each isolate’s purified DNA and utilizing known sizes of nucleic acids to analyze the composition of the purified samples. DNA samples were electrophoresed through 0.8% agarose gel in 0.5X TAE, stained with ethidium bromide (EtBr) and photographed using an EtBr filter with the FluorChem Gel Doc System (Alpha Innotech). Any samples shown to contain a large amount of RNA were treated were RNase overnight and reevaluated. Discussion and Conclusion This is part of an ongoing study that began in 2006 to determine the prevalence of N. fowleri in the state of Kansas, which may suggest a potential health risk in contaminated waters. Earlier, Dr. Mullican and his students recovered five N. fowleri samples from Potter’s Lake, Lawrence, Kansas. The present study expands those initial numbers. Numerous amoeba isolates were recovered with water samples from five freshwater sources in northeast KS. Although amoeba with morphological and growth characteristics similar to those found in N. fowleri were only detected in 59 samples out of 122 (49%), we plan to analyze the DNA collected from all of the isolates. Analysis of each amoeba isolate will be done using the polymerase chain reaction (PCR) with primers specific to N. fowleri gene sequences. Additionally, PCR using primers that amplify the ITS region of the rRNA cistron will be performed to determine if new Naegleria spp. have been identified or not. In addition to the 122 DNA samples, we still have many stored (refrigerator) plates containing amoebae plaques that need further analysis. To our knowledge there have been no reports of PAM in Kansas to date, yet N. fowleri is most likely present in freshwater sources within the State. There have been reported incidences of PAM in some states that border Kansas, so it is likely that the amoeba is present in Kansas freshwater reservoirs. Future, planned mice studies in collaboration with Dr. Francine Marciano-Cabral (Virginia Commonwealth University, Richmond) will help determine the potential pathogenicity of any Kansas isolates that are identified as N. fowleri or have N. fowleri-like characteristics. The results of this ongoing study suggests the presence of other (non-Naegleria) thermotolerant amoeba species in waters frequented by recreation seekers like water skiers, swimmers, etc. Further tests are needed to discover whether these other samples contain amoebae species that are known to be present in Kansas waters, and if these species are pathogenic to humans. The presence of N. fowleri in Kansas also opens the possibility that previous cases of fatal meningitis with unknown etiology may potentially be PAM cases attributed to N. fowleri infection. Upon confirmation, we will investigate possible collaborations with medical centers to examine tissue samples from patients dying from undiagnosed meningitis-like illnesses. This study confirms what is observed worldwide, that despite the prevalence of N. fowleri in a water source, the probability of an infection leading to PAM remains extremely remote. References 1. Fowler, M., and R. F. Carter. (1965) Acute pyogenic meningitis probably due to Acanthamoeba sp. A preliminary report. BMJ 2(5464): 740-742. 2. Carter R. F. (1970) Description of Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and the experimental pathological changes induced by it. J. Pathol. 100:217-244. 3. De Jonckheere J.F. (2002) A century of research on the amoeboflagellate genus Naegleria. Acta Protozool 41:309-342 4. Weik, R. R., and D. T. John. (1977) Agitated Mass Cultivation of Naegleria fowleri. J. Parasitol. 63:868-871. Location Total water samples Total plaques Survived @42°C Flagellate observed Lake Shawnee 3 15 15 (100%) 11 (73%) Central Park Fishing Lake 2 11 9 (82%) 7 (64%) Clinton Lake 2 8 8 (100%) 2 (25%) Lake Perry 1 2 1 (50%) 0 Potter’s Lake 7 84 50 (60%) 39 (46%) TOTALS 15 120 83 59 (49%) 1Kbp DNA ladder CPBD002 CPAD003 CPBD006 CPAD001 CPAD002 LSCD002 LSCD001 LSCD005 LSAD002 LSBD001 1 Kbp DNA ladder LSAD001 LSCD006 LSCD004 LSBD002 LSAD003 LSBD004 LSCD003 LSAD004 LSAD005 LSBD003 1Kbp DNA ladder PLES007 PLDD014 LSAD005 CLBD001 PLDS001 PLED001 PLDD023 PLCD006 LSCD003 PLCF2.1A PLDD019 PLED008 PLDS022 CPAD002 PLDS024 LSBD004 CPBD002 PLDS014 CPAD003 LSCD002 1Kbp DNA ladder LSCD004 LSAD003 PLES001 1Kbp DNA ladder PLDS010 PLCD004 LSAD004 CPBD003 LSCD005 PLDS008 PLCS005 CPBD001 LSCD001 LSBD003 PLED002 Figure 3. Analysis of Genomic DNA from Amoeba Isolates via Agarose Gel Electrophoresis. 1Kbp DNA ladder CPAD001 PLCD007 LSBD001 PLAF4.3 LSAD001 CLAD002 LSAD005 PLES004 CPBD005 LSAD002 1Kbp DNA ladder PLDS016A PLCD001 PLDD007 PLDD012 CLAD003 PLDS013A PLCS001 PLDD016 1 Kbp DNA ladder PLDS012 PLES006 PLES009 CLAD003 PLDD003 PLED004A CLAD004B CLAD001 PLCS001 PLDS001 CLAD044A PLES008 PLCS002 PLDS022 1 Kbp DNA ladder PLDS016B PLDS023B LPBD001 PLES010 PLES002 PLDD018 PLED007 PLDD008 PLED002 PLDD004B PLED006 PLDD004A PLDD015 PLED008 PLAD002 PLED004B PLDD001 CLBD002 PLDS015 CLAD002 CLAD003 PLDD021 1 Kbp DNA ladder Figure 2. Growth Characteristics of Amoeba Isolates. A. Agar plate demonstrating an expanding amoebae plaque of a sample originating from Potter’s Lake. B. Encysted amoebae C. Amoebae in the trophozoite (vegetative) stage. D. Growth of amoebae in liquid culture. cysts trophozoites Introduction The first case of primary amoebic meningoencephalitis (PAM) was observed in 1965 by M Fowler and R.F. Carter in Australia (1). This disease affects the central nervous system and has been found to be fatal to most all people infected by the parasitic organism known as Naegleria fowleri. The amoeboflagellate was identified and portrayed by Carter in 1970 (2). Naegleria spp. are ubiquitous in the environment and unidentified N. fowleri certainly poses a potential threat in many freshwater reservoirs. PAM is very rare and very few incidents are recorded each year world-wide. The severity of this disease is great; out of the two hundred or so reported cases since 1965, only two individuals have been known to survive following a vigorous treatment regimen (3). Surprisingly, the majority of individuals that have been effected by this disease-causing organism have been healthy (immunocompetent), but have had recent contact with warm sources of freshwater. N. fowleri is a particular hazard because of its thermotolerant nature. The organism has the ability to thrive in freshwater of temperatures up to 45°C (3). Another interesting characteristic of N. fowleri is it’s tri-cycle lifecycle. The amoeboflagellate harnesses the ability to morph between three distinctive stages: trophozoite (vegetative), cyst (dormant), and flagellate (temporary) forms (Figure 1). Of the three different forms, only the trophozoite (amoebae) and flagellate forms are thought to be infectious. Water samples were obtained in the summer of 2009 from five freshwater reservoirs in northeast Kansas: Lake Shawnee, Lake Perry, and Central Park Fishing Lake (Topeka), Clinton Lake and Potter’s Lake (Lawrence). The samples were cultured and assessed according to the previously mentioned morphological characteristics. This processing was utilized to select for isolates likely to be N. fowleri. These selected thermotolerant amoeba were then subjected to N. fowleri specific polymerase chain reaction (PCR), to locate species specific genetic markers that would definitively identify the existence of these deadly amoeba in local environments. Table 1. Summary of amoebae collected from five different freshwater sources. All of the amoebae grew at 37˚C. A. D. E. B. C. trophozoites A. B. C. D.