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Extended-spectrum beta-lactamase-producing Enterobacteriaceae in Flat Creek
Water Reclamation Center.
Lindsay McCuen
Abstract Extended-spectrum beta-lactamases (ESBLs) are enzymes that are capable of degrading frequently used beta-lactam antibiotics such as penicillin and cephalosporins. Organisms that produce these enzymes are often multi-drug resistant. ESBLs are commonly produced by the Enterobacteriaceae group of gram negative bacteria that are naturally occurring gut microbiota. Currently, ESBLs pose a significant health threat due to their ability to cause a multitude of difficult to treat infections, and are one of the leading causes of death world-wide. The bla genes that code for ESBL can be passed via horizontal transmission. As of yet, there is no data on prevalence of ESBL-producing Enterobacteriaceae in North Georgia water sources. Flat creek is located in North Georgia and runs directly into Lake Lanier, which is a main source of drinking water for the Atlanta area as well as a popular spot for water recreation. We obtained efferent and afferent water samples from Flat Creek Water Reclamation Center in Hall County Georgia. These water samples were filtered, grown in an enrichment media and plated on selective media that yielded the isolation of ESBL-producing Enterobacteriaceae. We found ESBL producing enterobacteria in all the samples we tested. Further identification methods revealed the presence of ESBL producing Klebsiella pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Citrobacter freundii and Escherichia coli. Following identification, disk diffusion method was used to test susceptibility of the isolates to several commonly used antibiotics. This testing revealed that all isolates were multi-drug resistant. Lastly, DNA was extracted from the isolates
to confirm the presence of bla genes that encode for ESBLs by agarose gel electrophoresis. Forty-two percent of the isolates were confirmed to be carrying one or more of the bla genes. Introduction Antibiotics are essential in modern medicine for fighting infectious diseases, and are vital to the survival of vulnerable patient populations with weakened immune systems. The overprescribing and misuse of antibiotics across human and animal populations has led to selection of antibiotic resistant bacteria (ARB). The consistent antibiotic pressure placed on bacteria facilitates ARB “superbugs” to thrive and flourish. (Chen et al., 2010). When common antibiotics become ineffective, increasingly toxic antibiotics are often the only option. Patients who acquire resistant infections face much higher morbidity and mortality rates. ESBLs are one such mechanism for bacteria to confer resistance. Extended-spectrum beta-lactamases (ESBLs) are enzymes that hydrolyze extended-spectrum cephalosporins with an oxyimino side chain, cleaving the beta- lactam ring, inactivating the antibiotic. The mechanism by which this occurs is rearrangement of the amino acid sequences of the active sites. (Patterson., 2003) Since first identified in 1983, ESBLs have become increasingly prevalent because ESBL enzymes can be plasmid mediated, thus their resistant genes are easily transferred between bacterial strains via horizontal and vertical gene transmission. Resistances to β-lactams mediated by extended-spectrum β-lactamases (ESBL) are especially relevant among Enterobacteriaceae (Ojer-Usoz, 2014). Antibiotics are essential in modern medicine for fighting infectious diseases, and are vital to the survival of vulnerable patient populations with weakened immune systems. The overprescribing and misuse of antibiotics across human and animal populations has caused the selection of antibiotic resistant bacteria (ARB). This consistent antibiotic pressure placed on
Sampling Sites and Water Sample Collection Between August 2015 and March of 2016, water sampling was conducted at Flatwater Creek Water Reclamation Facility in Gainesville, Georgia. We collected 100 mL of both efferent and afferent samples from the plant on August 2015, September 2015, and March 2016. 100 ml of water samples were collected from approximately 50 cm below the water surface using sterile bottles. The collected water samples were stored on ice for 30 minutes for transport to the lab. Microbiological Analysis For each sample, 100 mL of water was filtered through sterile 0.45 μm membrane filters (Nalage, Rochester, NY, USA). The filters were removed and placed in 20 mL of enterobacteria enrichment (EE) Broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator at 275 rpm for 24 h at 37 ºC. After incubation, a loop full of culture was streaked on an ESBL chromogenic Agar plates (Hardy Diagnostics, Santa Maria, CA) and incubated at 37°C for 24 hours. The chromogenic plates contain a proprietary blend of antibiotics to select for multi-drug resistant strains and allow for easy identification of E.coli and Kelbsiella via the chromogens (pink and blue respectively). The colonies that grew on the chromogenic plates with different colors and morphologies were isolated, sub-cultured, and confirmed for multi-drug resistance. The antibiotic resistant enterobacterial species were identified by using API strips (Biomeriux) as per the manufacturer’s instruction. Antimicrobial Susceptibility Testing and ESBL Confirmation We selected these specific antibiotics to be representative of increasing levels of resistance within the beta-lactam structured drugs as well as non-beta-lactam drugs. The higher the
generation, the more toxic the antibiotic. We used a first, second, third and fourth generation beta-lactams(Penicillin, Cefepime, Cefotaxime, Ceftazidime, Imipenem) as well as we used three beta-lactams with clavulanic acid (Augmentin, Ceftazidime/Clavulanic Acid, Cefotaxime/Clavulanic Acid) a known beta-lactamase inhibitor. We also used four non-beta- lactam antibiotics, (Chloramphenicol, Gentamycin, Tetracycline, Minocycline) that are common front line drugs. The disk diffusion method was used to test susceptibility of the isolates against 12 anti-microbial agents. The tested antibiotic concentrations: Cefotaxime-Clavulanic Acid 30/10μg, Cefotaxime 30 μg, Ceftazidime-Clavulanic Acid 30/10μg, Cefepime 30μg, Chloramphenicol 30 μg, Imipenem 10μg, Minocycline 30μg, Tetracycline 30μg, and Gentamicin 120μg. Isolates showing resistance to three or more antibiotic classes were defined as multidrug resistant (MDR). Polymerase Chain Reaction (PCR) to Detect bla Genes We extracted genomic DNA from the bacterial isolates using a DNA extraction kit (Thermo Scientific). We performed PCR to detect blaTEM, blaCTX-M, blaSHV, and blaOXA genes using specific primers found in previously published work (Chen et al., 2010). We used a 1μg genomic DNA, 2.5 μL of forward primer, 2.5 μL of reverse primer, 19μL of sterile water, and one PCR bead (GE healthcare, UK), for a total reaction volume of 25μL Results Variety of ESBL-Producing Enterobacteriaceae Identified. Multidrug resistant ESBL producing enterobacteria were found in both the efferent and afferent samples obtained in all three trials. 26 individual isolates were found to include Ent. coloacae (3), E. coli (5), Kl. Pneumoniae (9), Ent.
bla Genes Present in Isolates The bla gene is a plasmid encoding gene which codes for the production of an enzyme called beta-lactamase. Beta-lactamases come in four variations classified as types A, B, C, and D. The bla gene has been identified in four forms the TEM, SHV, CTX, and OXA genes. We found that 38.5% of all the isolates identified were positive for one of the four possible bla type gene. Of those, 50% of them were positive for two or more genes. Bla Gene Type Number of isolates containing the gene Percentage of Isolates SHV 4 15.4% CTX 5 19.2% OXA 3 11.5% TEM 5 19.2% Discussion In this study, 26 individual isolates of ESBL-producing Enterobacteriaceae were found in both efferent and afferent water samples from the Flat Creek Water Reclamation Facility in Gainesville, Georgia. This facility treats water that comes from industrial, healthcare, and residential waste. The ESBL Enterobacteriaceae that we found were all multi-drug resistant. They showed increasing resistance from the penicillin based antibiotics up through the third and fourth generation cephalosporins, as well as, resistance to the carbapenem. We also had
resistance to the non-beta-lactam antibiotics showing that these isolates have developed more than one mechanism of resistance as beta-lactamase would have no effect on a drug without a beta-lactam ring in its structure. This suggests that these strains have been subjected to multiple selective pressures being in the constant presence of antibiotics, thus causing them to become resistant to higher order drugs. There were some limitations in this study: There were only three trials performed over the course of a year which may not be representative of the entire picture. One of the trials was done during February, which turned out to be a very cold day, and that is the reason we believe we got far fewer isolates out of that trial. We have given but a snapshot of the entire issue concerning ESBL in our community. In future studies we plan to look at the correlation between anthropological impact and the species and enumeration of ESBL producing bacteria. We also plan to look at possible seasonality to explain the reasoning for the winter data being significantly less than spring/summer. ESBL Research References Abdel-Moein, K. A., & Samir, A. (2014). Major article: Occurrence of extended spectrum β– lactamase-producing Enterobacteriaceae among pet dogs and cats: An emerging public health threat outside health care facilities. AJIC: American Journal Of Infection Control, 42796-798. doi:10.1016/j.ajic.2014.03. Al-Bayssari, C., Dabboussi, F., Hamze, M., & Rolain, J. (2015). Detection of expanded-spectrum β-lactamases in Gram-negative bacteria in the 21st century. Expert Review Of Anti-Infective Therapy, 13(9), 1139. doi:10.1586/14787210.2015.
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