Dissemination of extensively drug-resistant and KPC-2 producing Klebsiella pneumoniae isolated from bloodstream infections

Introduction: Bloodstream infections (BSIs) are serious diseases associated with high mortality, especially when caused by extensively drugresistant (XDR) Klebsiella pneumoniae. The prevalence and pandemic strains of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae isolated from blood cultures of patients with BSIs were determined at Sir Run Run Shaw Hospital, China. Methodology: A total of 24 XDR K. pneumoniae were isolated from blood cultures, and the clinical data of the patients were analyzed retrospectively. Bacterial species identification and antimicrobial susceptibility testing were performed using VITEK2 and E-test methods, respectively. Common ESBL-resistant genes were amplified and sequenced after the validation of a modified Hodge test. Strain homology was also analyzed by pulsed-field gel electrophoresis (PFGE). Results: All of the isolates were resistant to 10 antimicrobial agents. Several strains showed partial sensitivity to aminoglycosides, but all showed sensitivity to polymyxin and tigecycline. All strains were Klebsiella pneumoniae carbapenemase (KPC-2) producing, and carried two or three ESBL-resistant genes, which belonged to 13 PFGE clones (A–M). The overall mortality rate in patients was as high as 29.2%. Conclusions: KPC-2-producing K. pneumoniae BSIs are associated with high mortality rates. Our observations suggest that KPC-2 and ESBL-producing K. pneumoniae might be responsible for the clonal dissemination of extensively drug-resistant isolates in our hospital.


Introduction
Bloodstream infections (BSIs) caused by Klebsiella pneumoniae carbapenemases (KPC)producing K. pneumoniae are associated with high mortality rates.[1].The isolation rate of resistant bacteria in BSIs has increased significantly with the rampant use of beta-lactams and aminoglycoside antibiotics, as well as the development of more invasive procedures and treatments [2].The Chinese Ministry of Health National Antimicrobial Resistance Investigation Net annual reports of 2010 and 2011 estimated that the isolation rate of K. pneumoniae, which has become one of the most common pathogenic bacteria in BSIs, was 7.7% and 8.3%, respectively [3,4].In this study, a total of 252 K. pneumoniae isolates were recovered from the blood of patients at Sir Run Run Shaw Hospital, a tertiary hospital, over the past two years; of the isolates, 24 were extensively drug-resistant strains.We investigated the prevalent characteristics of extensively drug-resistant (XDR) K. pneumoniae isolates in BSIs, which were resistant to almost all 15 antimicrobial agents tested.We also tested the βlactamase genotypes, examined the clonal relatedness of the strains, and analyzed the clinical data of patients to provide evidence for nosocomial infection.

Bacterial strains
From August 2010 to December 2012, clinical non-duplicate, consecutive K. pneumoniae isolates were recovered from BSIs of inpatients at Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University.Bacterial species identification was performed using the VITEK2 system (BioMérieux, Marcy l'Etoile, France), and the identified XDR strains were included in this analysis [5].The clinical data of these patients were also collected and analyzed.

Instruments and reagents
Both the VITEK2 system and the E-test method (AB bioMérieux, Solna, Sweden) were used to investigate the antimicrobial susceptibility profile.
CHEF pulsed field gel electrophoresis (PFGE), polymerase chain reaction (PCR), and gel image analysis systems were applied (all from Bio-Rad, Hercules, USA).A PCR amplification kit and the endonucleases ApaI and XbaI (TAKARA Bio Inc., Kusatsu, Japan,) were used to amplify common βlactamase genes for the modified Hodge test (MHT)positive strains.

PCR amplification of β-lactamase and DNA sequencing
PCR amplification of common β-lactamase genes was performed for MHT-positive strains.The amplified genes included bla KPC , bla IPM , bla VIM , bla SPM- 1 , bla GIM , bla SIM-1 , bla NDM-1 , bla OXA-48 , bla TEM , bla SHV , bla CTX-M (bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-8 , bla CTX-M-9 ), bla DHA , bla MIR , bla ACC , and bla CIT .Bacterial DNA template was extracted by the boiling method according to the previously reported protocol [6].PCR reactions were designed as follows: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 40 seconds.PCR products were confirmed by 1% agarose gel electrophoresis, and images were acquired under a UV lamp.Positive PCR products were sent to Bo Shang Biotechnology Co. for DNA sequencing.The sequence analyses were performed using the BLAST program available on the National Center for Biotechnology Information server (http://blast.ncbi.nlm.nih.gov/).

Pulsed-field gel electrophoresis (PFGE)
The clonal relatedness of the isolates was determined with PFGE, as described previously [6].A suspension of 0.5 McFarland bacteria was embedded in agarose and digested with XbaI for 4 hours at 37°C in advance.PFGE conditions were as follows: 6 V/cm 2 for 22 hours, with an initial pulse time of 5 seconds and a final pulse time of 25 seconds.PFGE patterns were read according to criteria defined by Tenover et al. [7] as follows: indistinguishable, variation in 1-3 bands; closely related, variation in 4-6 bands; and unrelated, variation in 7 or more bands.The phylogenetic tree was constructed by BioNumerics software version 7.0.

Demographic and clinical characteristics of the patients
In total, 24 XDR K. pneumoniae isolates associated with BSIs were isolated from the following departments: intensive care unit (ICU) (n = 11), hematology (n = 4), gastroenterology (n = 3), infectious diseases (n = 1), general surgery (n = 3), and urology (n = 2).Nineteen patients were male, and five were female.Patients' ages ranged from 30 to 84 years (mean age, 61.3 ± 14.2 years), and 16 patients were 60 years of age or older.A total of 4 patients were diagnosed with malignant tumors; 4 with leukemia or other blood diseases; 8 with cholecystitis and other diseases of the digestive system; 2 were cases of trauma; and 6 had hypertension, diabetes, or other chronic diseases.A total of 19 patients received catheter intervention, and 24 patients received betalactamase antibiotics before the isolation of K. pneumoniae.Fourteen days after being infected, 17 patients were alive and 7 had died; the mortality rate was 29.2%.The clinical and demographic information of the patients are listed in Table 1.

Antimicrobial susceptibility results
Antimicrobial susceptibility tests for 15 antibiotics showed that all of the isolates were resistant to penicillin, cephalosporins, penicillin or penicillin enzyme inhibitors, aztreonam, and fluoroquinolones.Three strains were susceptible to aminoglycoside antibiotics, tobramycin, and amikacin.All 24 isolates were sensitive to polymyxin and tigecycline, and the results are listed in Table 2.The MICs were interpreted according to the 2013 CLSI guidelines.

Discussion
Previous studies have indicated that long-term use of catheters is the main cause of BSIs, and that the frequent use of antibiotics such as carbapenems is one of the main risk factors associated with antimicrobial resistance acquisition in K. pneumoniae [8].The retrospective analysis in our study showed that among 24 cases, 9 cases had malignant disease; 6 cases had digestive disease; 7 cases had hypertension, diabetes and various types of chronic disease; and only 2 cases were of traumatic injury.Before the isolation of XDR K. pneumoniae, most of the patients had been exposed to invasive procedures, and all of the patients had received β-lactamase antibiotics.The elderly accounted for the majority of our patients, most of whom had chronic diseases.It is reasonable to postulate that due to their weakened immune systems, exposure to antimicrobial therapy, and invasive devices, these patients are likely to have significantly different risk factors compared to normal patients [8,9].With these confounding variables, patients infected with multi-drug resistant bacteria also have an increased mortality risk [10].The 14-day mortality for patients with drug-resistant K. pneumoniae BSIs has been reported to be up to 41.7% [11].The overall mortality in our patients was high (29.2%),similar to that observed in other studies [10,11].
Carbapenems have been widely utilized as the treatment of choice for serious infections caused by ESBL producers, exerting selection pressure for carbapenem resistance acquisition [5].Carbapenemresistant K. pneumoniae, especially KPC-2-producing isolates, have been therefore disseminated globally and pose remarkable clinical and therapeutic challenges [12].The increasing prevalence of severe infections with high mortality rates underlines the need for effective treatment.In our study, antimicrobial susceptibility tests showed that all of the 24 KPC-2 positive isolates were extensively drugresistant and completely resistant to penicillin, cephalosporins, penicillin/penicillin enzyme inhibitors, aztreonam, and fluoroquinolones.These strains were highly resistant to carbapenems, but only susceptible to polymyxin and tigecycline, which have been ultimately considered as the last-resort treatment for such infections.Previous research demonstrated that antimicrobial-resistant K. pneumoniae isolates could carry multiple resistance genes simultaneously [13,14].In our study, a high prevalence of β-lactamase genes was observed.All of the strains carried bla TEM-1 , 21 carried bla SHV-11 or bla SHV-12 , and 21 carried bla CTX- M .Among bla CTX-M carriers, 15 strains carried bla CTX-M- 24 , indicating that bla CTX-M-24 was the major epidemic type of bla CTX-M , and 3 strains carried bla DHA-1 .All of the strains carried multiple resistance determinants in various combinations, especially the combination of bla TEM-1 , bla SHV-11 , and bla CTX-M-24 .According to previous studies both in China and the United States of America (USA), the most commonly identified bla CTX- M type was bla CTX-M-15 in K. pneumoniae [15,16].However, this study reports, for the first time, that in our hospital, bla CTX-M-24 is the major type of bla CTX-M and should be monitored with care.PFGE analysis showed that 24 XDR K. pneumoniae BSIs in our hospital had 13 clonal clusters (A--M).Eleven isolates in the ICU were from 9 clusters; only 2 strains were type E, 2 were type G, and the remaining 7 strains had different spectral types.Moreover, J, K, L, and M types appeared only in the ICU, and the other 5 types were detected in other departments.This distribution could be primarily due to the high frequency of transfer of critically ill patients in the ICU ward.Patients are transferred to the ICU when they are critically ill, and they are transferred back to their designated ward when they are in stable condition.Therefore, the ICU has become an important route of transmission by which bacterial strains are embedded and then disseminated to other patients.Strains are then carried to other departments with the patients as they recover and leave, or as they are transferred between units for specialized procedures or treatment.In our study, the clonal spread existed not only between the ICU and other departments, but also among other departments.For example, strain No. 224 was isolated from a patient with ampullary carcinoma in the department of general surgery, and strain No. 225 was collected from a patient with obstructive jaundice in the gastroenterology department.Patient No. 224 had an endoscopic retrograde cholangiopancreatography (ERCP), and patient No. 225 also received the same procedure.Interestingly, D clonal type XDR K. pneumoniae could also have been isolated from both departments.Thus, it is reasonable to speculate that the use of medical devices is another risk factor that causes XDR K. pneumoniae transmission among different departments.Therefore, we concluded that simultaneous sporadic and clonal dissemination were responsible for the wide distribution of XDR K. pneumoniae isolated in multiple departments of our hospital.

Conclusions
It is evident that most of the BSI patients with XDR K. pneumoniae had malignant and chronic diseases with high mortality.The XDR K. pneumoniae isolates harbouring bla KPC-2 also carried several ESBLresistant genes, and the strain spread between the ICU and other departments in both was both sporadic and clonal.Screening and surveillance of XDR K. pneumoniae is urgently needed in our hospital to control and prevent the further spread of these resistance genes and resistant organisms.

Figure 1 .
Figure 1.PFGE analysis and β-lactamase genes of the 24 K. pneumoniae strains of bloodstream infections

Table 1 .
Demographic and clinical characteristics of the 24 patients with extensively drug-resistant K. pneumoniae strains of bloodstream infections ICU: intensive care unit; PICC: peripherauy inserted central catheter; COPD: chronic obstructive pulmonary disease