As effective as antibiotics have been since they were first discovered, antibiotic resistance proves to be a worldwide threat, endangering their efficacy. Over the years, there has been a significant decline in development of novel class antibiotics. These two factors have made once easily treated infections difficult to manage. Carbapenem‐resistant Enterobacteriaceae (CRE) are an emerging threat due to their high levels of antibiotic resistance. CRE have become resistant to almost all antibiotics, and cause 23,000 infections per year in the United States. Metals are a vital nutrient in all living organisms including bacteria and, have thus been targeted by antimicrobial agents. Bacteria, like all living organisms, must regulate their metal homeostasis in order to survive and replicate. Therefore, mechanisms able to diminish the availability of the metals to bacterial pathogens can be used to develop new therapeutic drugs. An example of a mechanism of bacterial resistance to antibiotics is the production of inactivating enzymes, such as carbapenemases, an example of which is seen in Klebsiella pneumoniae. Carbapenemases play an important role against b‐lactam antibiotics such as imipenem in Enterobacteriaceae. Carbapenemases are categorized based on their functional and molecular properties. Class B carbapenemases are metallo‐b‐lactamases (MBL) which require zinc in their active site, and are therefore considered metalloenzymes. MBL can hydrolyze all b‐lactams and are not inactivated by b‐lactamase inhibitors. In this study we analyzed antimicrobial susceptibility of K. pneumoniae isolates, KPC 2146, KPC 1705 and KPC 1706 which contain class B, class A and no carbapenemase, respectively. Metal homeostasis of isolates was disrupted by inducing zinc deficiency using TPEN, a metal chelator with high affinity for zinc, and preparing a zinc deficient minimal media. Carbapenemase activity was assessed using CARBA‐NP test and LC‐MS. Bacterial susceptibility of zinc‐deficient isolates to carbapenems was analyzed using spot plating, growth curve, minimum inhibitory concentration and ring of inhibition. Our findings demonstrate zinc dependency of class B carbapenemase in the KPC 2146 isolate, while class A carbapenemase in the KPC 1705 isolate was not affected. Spot plating and growth curve analyses demonstrated that zinc deficiency lowers bacterial growth in KPC 2146 in the presence of imipenem. Zinc deficiency via TPEN addition lowered imipenem, doripenem, ertapenem and meropenem MIC in KPC 2146. Ring of inhibition of KPC 2146 with imipenem was greatly affected by zinc chelation. In conclusion, we demonstrate that disrupting zinc homeostasis affects class B carbapenemase activity, which in turn increases bacterial susceptibility to carbapenems. Support or Funding Information Nika Padyab was supported by funds from the Biomedical Sciences Program at MWU. Drs. Veltri and Hernandez were supported by MWU intramural funds.
Klebsiella is a genus of bacteria frequently associated with nosocomial infections due to their multi‐drug resistant properties. Klebsiella pneumoniae accounts for a large majority of hospital‐acquired pneumonia, urinary tract infections, and septicemia. The pathogenicity of K. pneumoniae clinical isolates has been greatly established to better understand and treat these infections. Klebsiella oxytoca is also primarily healthcare associated and is considered to be an emerging nosocomial pathogen, as it causes the second most frequent infections from the Klebsiella genus. Although it shares many characteristics with K. pneumoniae, its virulence factors have not been well studied due to its lesser degree of incidence. As an emerging pathogen, more information must be gathered in order to learn how to best treat this infectious, antibiotic‐resistant bacteria. This study aims to first determine if the pathogenic factors found in clinical Klebsiella pneumoniae isolates are also present in Klebsiella oxytoca and then compare the production of these virulence factors under normal and inducing conditions. This work compares the expression of pathogenic factors in five isolates of K. oxytoca, three isolates of K. pneumoniae, and one strain of Escherichia coli. The pathogenic factors chosen for this study include antibiotics resistance, siderophore production, biofilm formation abilities, and reactive oxygen species (ROS) detoxification. To determine antibiotic resistance, the minimum inhibitory concentration of nine different antibiotics was calculated for each isolate. Siderophore production was measured using a Chrome Azurol Sulfonate colorimetric test in both the absence and presence of 2,2’‐dipyridyl, an iron chelator. Biofilm formation was detected with Crystal Violet dye in both the absence and presence of acetaminophen and hydrochlorothiazide, both of which are commonly used therapeutic pharmaceuticals in nosocomial settings, as well as 2,2’‐dipyridyl. ROS detoxification via superoxide dismutase enzyme activity was measured through native gel activity assays. Our findings indicate that K. pneumoniae has resistance to more antibiotics than K. oxytoca, but the two species have comparable siderophore production, biofilm formation capabilities, and ROS detoxification capabilities. In conclusion, Klebsiella pneumoniae and Klebsiella oxytoca share several mechanisms of pathogenicity under normal and inducing conditions and drug resistance is the most notable difference among the strains tested in this work.
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