Carbapenem-Resistant Enterobacteriaceae Co-Infections with Klebsiella ; a Retrospective Study

Background: Carbapenem antibiotics are often used to treat bacterial infections that are refractory to other more commonly used antibiotics. The resilience of bacteria and their resistance mechanisms are of novel interest in the clinical setting, as antibiotic resistant bacteria are posing a challenge to health care professionals. The goal of this retrospective chart review was to isolate Carbapenem resistance among co-infections of Klebsiella and other microorganisms and to conduct a systematic review of this antibiotic resistance.


Introduction
The use of carbapenem antibiotics such as imipenem and meropenem has increased significantly in the past several years.This has been due to increased antibiotic resistance of many species of bacteria in the hospital setting associated with acquisition of one or more resistance mechanisms.Carbapenem antibiotics are often used to treat bacterial infections that have become refractory to antibiotic treatment attempts using other classes of antibiotics such as cephalosporins and aminoglycosides.
Recently, bacteria such as Klebsiella, E. coli, and E nterobacter have developed resistance to carbapenem antibiotics in the hospital setting.These types of microbes have been designated as carbapenemresistant Enterobacteriaceae (CRE) [1].
CRE is becoming a major problem for hospitalized patients.As speculated by Goren et al., even for healthy individuals, CRE can often outcompete normal gut flora and therefore colonize the gut, which can become a source of infection for patients [1,2,3].
The mechanisms by which Enterobacteriaciae acquire and spread resistance to carbapenem antibiotics have become a point of interest in current research.Mechanisms for transferring or developing resistance include plasmid transfer from species of CRE such as Klebsiella to other microorganisms, decreased porin expression in the outer membrane, and genetic mutations have been proposed as mechanisms of resistance [4].
The focus of this retrospective chart review was on patients who had a co-infection with carbapenem resistant Klebsiella and another microorganism, which ultimately acquired resistance to carbapenem, presumably by a resistance transfer mechanism.

Methods
This study involved a retrospective, single center chart review of patients that were admitted to Kingsbrook Jewish Medical Center from June 2011 to June 2014, who had positive cultures of both E. coli and Klebsiella.Resistance to carbapenam was reviewed on the microbiology report in the patient's chart, and a timeline was constructed from the initial infection to resolution of infection, with special reference to the presence or change in carbapenem resistance.There was no distinction based on site of infection.

Results
Based on patient chart analysis, two cases were found with Klebsiella and E. coli co-infection with carbapenem-resistant strains.Both patients were admitted from nursing home.Patient 1 had a coinfection of Klebsiella and E. coli in a wound.Initially, Klebsiella was carbapenem resistant, while E. coli was carbapenem susceptible.One week later, when the wound culture was done again, both Klebsiella and E. coli were carbapenem resistant.Months after, the patient developed a Klebsiella infection in the urinary catheter that was carbapenem resistant.The patient had co morbidities such as living in a nursing home and having an indwelling catheter.
Patient 2 had an initial urinary tract infection with Klebsiella that was carbapenem resistant.Two weeks later, the patient was found to be co-infected with Klebsiella and E. coli.that was carbapenem resistant.A clean catch urine specimen was conducted two weeks later, and only E. coli was carbapenem resistant while Klebsiella was carbapenem sensitive.

Discussion
One can speculate that in patient 1, the E. coli acquired carbapenem resistance transfered from Klebsiella.Urban et al. demonstrated the presence of E. coli harboring Klebsiella carbapenemase betalactamases as a result of plasmid transfer from Klebsiella to E. coli [5,6].In this scenario, Klebsiella would carry the genes titled KPC-2 and KPC-3, both of which contribute to Klebsiella being resistant to carbapenem [7].Interaction between Klebsiella and E. coli would enable plasmid transfer, and would confer the same carbapenem resistance to the E. coli.
Another proposed mechanism of E. coli developing carbapenem resistannce would not involve Klebsiella gene transfer, but genetic mutations to the porin on the outer membrane of E. coli.It has been demonstrated that Enterobacteriaceae species, such as E. coli, can undergo genetic mutations in genes such as OmpF and OmpC, which are responsible for porin production [7,8].Lack of porin production is associated with resistance to carbapenem, as the drug has no mechanism of crossing the cell membrane and gaining access into the bacteria [8,9,10].
Silencing of the marRAB operon in Enterobacteriaciae species can also be proposed as a means of carbapenem resistance.The mar operon is also linked to porin expression.Studies by Chollet et al. demonstrate that this operon is influenced by the presence of antibotics [11,12,13].The mar operon is regulated by marA and marR, with marA inducing antibiotic resistance and marR, which down regulates antibiotic resistance [11,12,13].MarA and marR are influenced by several factors, one of them being the bacteria's environment.If the bacteria is subjected to antibiotics, marA can be activated to induce resistance, while an environment without antibiotics would enable marR to be activated and down regulate antibiotic resistance [11,12,13].It can be noted that while Klebsiella and E. coli share a similar genomes in regards to the mar operon, it is uncertain if factors from Klebsiella can influence the genome of E. coli [14].
Patient 1 had a long term wound infection that spanned several months.The patient was initially infected May 6, 2011 and continued to be infected until August 22, 2011.It should be noted that in the month of August, the patient's urinary catheter tested positive for Klebsiella that was resistant to carbapenem.One can speculate that long-term infection of the patient's wound would put the patient at a high risk for a secondary infection-in this case a catheter infection.Bisson et al. speculate that an important risk factor for developing an infection with bacteria exhibiting extended antibiotic resistance properties is hospital stay [14].Moreover, the CDC also supports this notion as the majority of cases of CRE are in patients who are in long-term care facilities [16].
Since the patient was in a nursing home, the individual would be at a greater risk of acquiring a secondary infection.As reported by CDC, the rate of CRE in a nursing home is 30%.Lowe et al. speculate that long term care facilities, such as nursing homes, have yet to develop standard protocol for treating patients that have CRE [17,18].Failure to consistently implement standard protocols for treatment and implementation of contact isolation precautions, puts individuals at risk for developing a secondary CRE infection or spreading the CRE infection to other residents.
The medical timeline for patient 2 is presented below (Figure 1).The medical chart showed that there was an initial clean catch urine obtained on 4/5/13 which grew only Carbapenem resistant Kle- bsiella, A second urine collected on 4/16/13 showed both carbapenem resistant E coli and Klebsiella.On 5/8/13 a third urine culture grew carbapenem resistant E coli and carbapenem susceptible Klebsiella.Chart review revealed the patient did not have any fever while under hospital care and no elevated WBC One can only speculate why Klebsiella lost resistance to carbapenem as evidenced by the culture results on 5/8/13.A possible explanation would relate to the marRAB operon and how it is regulated.The marRAB is influenced by the presence of antibiotics [11,12,13].Initially, when the patient had an E. coli and Klebsiella infection, the patient was receiving no antibiotic treatment and both species were carbapenem resistant.It can be hypothesized that after several weeks of no antibiotic treatment, there was induction of marR that would silence the marRAB sequence, which would result in increased porins, and therefore, increased susceptibility to carbapenem [19,20].One can speculate as to why Klebsiella lost resistance to imipenem and E. coli still retained resistance.The timing of gene regulation can provide an explanation for this.For instance, Klebsiella may induce marR quicker than E. coli so it would lose resistance at a faster rate and therefore be more susceptible earlier.The method of gene regulation in bacteria can also provide an explanation to this.Induction of marR is regulated via a cascade.The mechanism of regulation of the marR cascade could be dependent on the bacteria species.Certain bacteria may regulate the gene cascade differently and therefore this would alter gene expression at varying rates.

Conclusion:
Co-infections with carbapenem resistant strains of Klebsiella and E coli are a point of interest in medical microbiology.The findings suggests that carbapenem-resistant Klebsiella can influence genetic traits (i.e.transfer of carbapenem resistance) of other En-terobacteriaceae such as E coli when present in the same environment or body site.
The phenomenon of carbapenem resistance transfer between different species of bacteria will likely cause more widespread carbapenem resistance and will greatly reduce the usefulness of some of potent antimicrobials Speculating that Klebsiella can down regulate certain genes can be of interest clinically.Rigorous antibiotic treatment can induce bacteria to up regulate resistance to antibiotics, and therefore, hinder resolution of an infection.Further investigation is needed to identify iatrogenic associations and causality of CRE in order to prevent further bacterial resistance and more importantly, spread of CRE.

Financial Disclosure
The Authors did not report any potential conflicts of interest.
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