Carbapenem resistance among the Enterobacteriaceae
is an emerging phenomenon of vast clinical and public health importance. Controlling the spread of KPC enzymes is difficult once the gene encoding this enzyme reside on transmissible plasmids
]. Current automated susceptibility testing methods have failed to reliably detect carbapenem resistance among K. pneumoniae
]. In this study, we obtained MICs for K. pneumoniae
isolates by broth microdilution, thus avoiding the misclassification of some case patients as potential control subjects. During the study period, all K. pneumoniae
isolates with MIC ≥ 2 μg/ml detected by the Vitek system were submitted for other susceptibility tests and screened for KPC production. K. pneumoniae
isolates harboring KPC enzymes had MICs for carbapenem in a range that allowed K. pneumoniae
to remain susceptible to carbapenem, and could therefore go unrecognized. Since 2010 CLSI have changed breakpoints and Enterobacteriaceae
isolates with MICs for imipenem and/or meropenem ≥ 2 μg/ml have been categorized as intermediate or resistant (CLSI, M100-S20-U). Therefore, some strains included in our study would be classified as imipenem and/or meropenem resistant if the most recent CLSI breakpoints are applied.
The presence of a premature stop codon in the porin gene could explain why some K. pneumoniae isolates included in the present study presenting an ompK35 or ompK36 amplicon size of 1000 bp are resistant to carbapenens.
During the 32 months of the study period, 20 patients were diagnosed with healthcare-associated infections caused by carbapenem-resistant K. pneumoniae
strains in our hospital. This represented 8.5% of the total episodes of K. pneumoniae
healthcare-associated infections. Analysis of data on infectious disease outcomes of patients revealed that carbapenem-resistant K. pneumoniae
patients had a higher mortality compared with patients infected with carbapenem-susceptible K. pneumoniae
(50.0% and 27.5%, respectively), although it was not statistically significant (p=0.085). Similar harmful effects on patient outcomes have been observed in previous studies where carbapenem-resistant K. pneumoniae
associated mortality was between 30 and 50%
]. These studies examined the epidemiology of KPC producers during K. pneumoniae
related-infections. Of interest, although patients included in our study were infected by carbapenem-resistant K. pneumoniae
strains that did not produce KPC carbapenemase, they had similar outcomes in terms of mortality.
Evaluation of the factors that predict carbapenem resistance by univariable analysis, demonstrated that prior ICU stay, central venous catheterization, longer use of a central venous catheter, and exposure to antimicrobials were associated with carbapenem-resistant K. pneumoniae
infection. In the multivariable analysis only the length of central venous catheter use was independently associated with K. pneumoniae
carbapenem resistance. Previous studies reported similar risk factors for carbapenem-resistant K. pneumoniae
infection and demonstrated associations with length of hospital stay, ICU admission, use of central venous catheter, recent solid-organ or stem-cell transplantation, receipt of mechanical ventilation, and exposure to broad-spectrum antibiotics
]. Overestimation of the importance of antibiotic exposure as a risk factor is a common selection bias in case–control studies in which control subjects have susceptible isolates. Surprisingly, in this study carbapenem use was not an independent predictor for carbapenem resistance. This unexpected finding may be related to the small sample size of this study.
To further explore the risk of mortality in K. pneumoniae-infected patients (both case studies and controls), we evaluated the impact of patient characteristics and treatment interventions. Unexpectedly, the initial treatment of patients with antibiotics for clinical isolates that were in vitro susceptible to treatment was not associated with patient survival.
Therefore, poor patient outcomes cannot be fully explained by a delay in providing the appropriate therapy. Previous studies have suggested that removal of the focus of infection, such as a catheter, debridement, or drainage, is an effective way of improving survival among patients with carbapenem-resistant K. pneumoniae
]. However, this adjunctive therapy was not evaluated in our study.
Besides observing clinical characteristics, we also performed molecular analysis of isolated K. pneumoniae strains to analyze the mechanisms of antimicrobial resistance, and to rule out the possibility of an outbreak during the study period. Although carbapenemase encoding genes including blaKPC were not identified in any of the K. pneumoniae isolates studied, blaCTX-M-2 and blaGES-1, ESBL encoding genes were detected in our collection. In addition, we observed changes in OMP-encoding genes amplicon size by PCR in 9 isolates suggesting the likelihood of altered porin functions. The amplicon size expected for ompK35 or ompk36 amplification is around 1000 bp. Through the DNA sequencing of some of these amplicons, we observed that acquisition of insertion sequences were responsible for the unexpected, higher molecular size of the ompK35 or ompk36 amplification. Therefore, we conclude that impermeability of outer membrane proteins contributed considerably to carbapenem decrease susceptibility in those K. pneumoniae isolates, especially when these isolates were ESBL producers (especially CTX-M-2- producing K. pneumoniae).
Most cases did not cluster in time and space. Molecular epidemiology revealed that the isolates belonged to seven distinct clones, although one subtype was predominant. However, most cases could not be linked to a specific patient-to-patient transmission event or to a common source.
Our results show that cephalosporinase production associated with porin modifications likely contributed to carbapenem resistance. This study focused on bacterial infection not colonization, and this allowed for a more accurate analysis of prognosis and mortality, as we only included patients with ongoing infections.
There were a number of limitations in this study. First, we had a low number of episodes of carbapenem-resistant K. pneumoniae infections in our hospital, suggesting the number of samples analyzed was small. Moreover this sample size may be underpowered to detect small significant differences. Second, we were not able to include prior colonization with carbapenem-resistant K. pneumoniae in our risk factor analysis for invasive infection, because the colonization status of each patient was unknown. At that time point we did not perform active surveillance for carbapenem-resistant Enterobacteriaceae (by rectal or peri-rectal swabs). Third, the case–control design for analyzing the risk factors for antimicrobial resistance has some limitations. The use of patients infected with carbapenem-susceptible K. pneumoniae as control subjects may be falsely inflated prior antimicrobial exposure (which was not observed in our study). The ability to match control-patients on important variables, such as time at risk and location, is problematic in case–control studies. Finally, because this study was performed at a single medical center, these results may not extrapolate to other hospitals.