Thursday, September 4, 2008

Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine

Schjørring, S., Struve, C., & Krogfelt, K.A. (2008). (e-publ. Aug. 13, 2008) Journal of Antimicrobial Chemotherapy doi: 10.1093/jac/dkn323.

Nosocomial infections, commonplace in health care systems including intensive care units (Çaatay et al., 2007), are becoming more of a pressing issue as bacteria continue to exhibit multiple antimicrobial resistances. Such cases have been reported in hospitals as well as extended care facilities such as nursing homes (Wiener et al., 1999). While the development of antibiotics are important in our fight against pathogens, it is equally important to focus on the mechanisms involved in developing increased resistance. In this paper by Schjørring, et al., one of the most common nosocomial pathogens was studied: Klebsiella pneumoniae, gram-negative bacteria shown to exhibit multiple antimicrobial resistances. The authors studied the effects of introducing antimicrobial genes and monitoring the colonization of K. pneumonia in mice intestines. The findings reveal several pieces of information about K. pneumonia, including the nature of the pathogen as well as its’ ability to transfer resistance genes to other bacteria (Schjørring, et al., 2008).

The authors of this article, created an intestinal colonizational model in order to observe this transfer more readily, so the plasmid transfer procedure was observed both in vitro and in vivo (Schjørring, et al., 2008). K. pneumoniae strain MGH75875 was used to follow the transfer process including colonization, and horizontal gene transfer (Schjørring, et al., 2008). This strain was originally isolated from an intensive care unit (ICU) patient with pneumonia. K. pneumoniae MGH75875 is currently known to be resistant to ampicillin, streptomycin, tetracycline, nalidixic acid, ticarcillin, trimethoprim/ sulfamethoxazole, cefotaxime and gentamicin, and is susceptible to imipenem (Schjørring, et al., 2008).

The plasmids monitored in this experiment presented interesting results on the basis of in vitro and in vivo examination. Several of the plasmids monitored (only named by their relative size) showed that environmental conditions do influence the nature of transfer. For example, the in vitro experiments showed transfer of the 108 or 157 kb plasmid, while in vivo only showed transfer of the 89 kb plasmid (Schjørring, et al., 2008).

The mice used in this study were individually caged and had unlimited access to resources, including food and water; antibiotics were administered through the water, at dosages described in the protocol. To begin, mice were first inoculated with the strain K. pneumoniae. This was done by growing up overnight cultures and resuspending the cultures in a 20% sucrose solution. Each mouse was given 100μL of this solution orally and subsequently their fecal matter was measured for bacteria; up to 109 cfu/g feces was found (Schjørring, et al., 2008). To determine the effects of antimicrobial treatment on the intestinal flora, three mice were treated with only K. pneumoniae and later treated with ampicillin added to their drinking water to represent treatment of infection. To determine the colonization of the intestine, two mice per experiment were treated with 0.5g/L ampicillin prior to exposure to the strain. Finally, to monitor gene transfer in the intestine three mice per experiment were treated with 0.5 g/L streptomycin sulphate in their drinking water prior to inoculation with the recipient strain as well as during the experiment (Schjørring, et al., 2008). A verification of transconjugants was done, via biochemical marker assays and by DNA isolation to provide a plasmid profile to detect the E. coli transconjugants. Also, a PCR was used to detect the presence of the plasmid-encoded extended spectrum β-lactamases (ESBL) genes, or the genes that code for antimicrobial resistance. In mice without any antimicrobial pretreatment, the inoculated strain quickly dropped below the detection limit due to the competitive nature of the other strains of bacteria present in the intestine. With the introduction of an antimicrobial treatment, there was an immediate increase in the population of the MGH75875 strain up to 109 cfu/g feces (Schjørring, et al., 2008). This experiment thus shows a direct relationship between selection factors and the immediate colonization of the gastrointestinal tract (GI) by the resistant pathogen (Schjørring, et al., 2008). There was also an observable higher transfer frequency of different plasmids into E. coli from K. pneumoniae during colonization of the mouse intestine. K. pneumoniae is thus an excellent colonizer in the GI tract of antibiotic-pretreated mice, and highly promiscuous with respect to numerous plasmids. The observed increase in the number of resistant bacteria, which can inherently lead to an increased risk of spreading resistance genes (Schjørring, et al., 2008).

After reading this paper I became very interested in the concept of evolution in our everyday lives. Most of us imagine evolution as a long and gradual process; however, in microbiology a normal 24-hour period can consist of several generations of bacteria. In this way, evolution can be easily observed and measured especially in the presence of selection factors. As a future physician, I recognize the importance of studying the relative effects of antibiotic use, including those associated with overuse, underuse and more recently the effects associated with resistant strains of bacteria in medicine. While studying antibiotic use is important, equally important is gaining a better understanding of what mechanisms are associated with resistance. Through this research and others, we all may come to appreciate what role evolution plays in our everyday lives, including our health care.

Related Articles of Interest:

Çaatay, A.A., Özcan PE, Gulec L, et al. Risk Factors for Mortality of Nosocomial Bacteraemia in Intensive Care Units. Med Princ Pract 2007;16:187-192.

Wiener, J., Quinn, J.P., Bradford, P.A., Goering, R.V., Nathan, C., Bush, K., Weinstein, R.A. JAMA 1999; 281: 517-523.

Nick Hardin, Undergraduate Researcher
University of Idaho

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