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Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 2016 Nov 21;60(12):7357–7363. doi: 10.1128/AAC.01503-16

Risk Factors, Outcomes, and Mechanisms of Tigecycline-Nonsusceptible Klebsiella pneumoniae Bacteremia

Chih-Han Juan a, Yi-Wei Huang b, Yi-Tsung Lin a,c,, Tsuey-Ching Yang b, Fu-Der Wang a,d
PMCID: PMC5119017  PMID: 27697759

Abstract

A rise in tigecycline resistance in Klebsiella pneumoniae has been reported recently worldwide. We sought to identify risk factors, outcomes, and mechanisms for adult patients with tigecycline-nonsusceptible K. pneumoniae bacteremia in Taiwan. We conducted a matched case-control study (ratio of 1:1) in a medical center in Taiwan from January 2011 through June 2015. The cases were patients with tigecycline-nonsusceptible K. pneumoniae bacteremia, and the controls were patients with tigecycline-susceptible K. pneumoniae bacteremia. Logistic regression was performed to evaluate the potential risk factors for tigecycline-nonsusceptible K. pneumoniae bacteremia. Quantitative reverse transcription-PCR was performed to analyze acrA, oqxA, ramA, rarA, and kpgA expression among these isolates. A total of 43 cases were matched with 43 controls. The 14-day mortality of patients with tigecycline-nonsusceptible K. pneumoniae bacteremia was 30.2%, and the 28-day mortality was 41.9%. The attributable mortalities of tigecycline-nonsusceptible K. pneumoniae at 14 and 28 days were 9.3 and 18.6%, respectively. Fluoroquinolone use within 30 days prior to bacteremia was the only independent risk factor for tigecycline-nonsusceptible K. pneumoniae bacteremia. The tigecycline-nonsusceptible K. pneumoniae bacteremia was mostly caused by overexpression of AcrAB and/or OqxAB efflux pumps, together with the upregulation of RamA and/or RarA, respectively. One isolate demonstrated isolated overexpression of kpgA. In conclusion, tigecycline-nonsusceptible K. pneumoniae bacteremia was associated with high mortality, and prior fluoroquinolone use was the independent risk factor for the acquisition of tigecycline-nonsusceptible K. pneumoniae. The overexpression of AcrAB and/or OqxAB contributes to tigecycline nonsusceptibility in K. pneumoniae.

INTRODUCTION

Klebsiella pneumoniae is an important cause of bacteremia worldwide. In one population-based study of K. pneumoniae bacteremia, the case fatality rate was 20%, and the annual population mortality rate was 1.3 per 100,000 (1). The rise of multidrug resistance (MDR) in K. pneumoniae harboring extended-spectrum β-lactamases or AmpC β-lactamases has resulted in the widespread use of carbapenem antibiotics (2). The increased use of carbapenem antibiotics as the last resort for the treatment of MDR K. pneumoniae (MDRKP) has accelerated the emergence of carbapenem-resistant K. pneumoniae (CRKP) (3). Tigecycline and colistin were introduced to treat infections caused by CRKP (4).

Tigecycline, a minocycline derivative, has in vitro activity against most MDRKP or CRKP (5). Tigecycline nonsusceptibility among K. pneumoniae isolates, as reported in recent studies (68), is between 0 and 5.7% globally (9). Recently, we reported on the high mortality rate (28-day mortality: 38.9%) in patients with tigecycline-nonsusceptible K. pneumoniae bacteremia (8). Although tigecycline-nonsusceptible K. pneumoniae bacteremia has emerged as a critical problem, studies reporting the risk factors for such infections are limited (6, 10). Indeed, as far as we can tell, no studies focusing on the risk factors for tigecycline-nonsusceptible K. pneumoniae bacteremia have been published in the scientific literature.

Tigecycline resistance in Enterobacteriaceae is attributed to resistance/nodulation/cell division-type pumps and to other efflux pumps (11). The RamA and AcrAB pathway and RarA, together with OqxAB pathways are implicated in tigecycline resistance in K. pneumoniae (12). Studies of tigecycline resistance mechanisms in K. pneumoniae usually include only a small number of isolates in the literature (1214). Moreover, studies addressing both the clinical features of bacteremia and the tigecycline resistance mechanism have never been reported.

In this study, we sought to identify the risk factors and outcomes for adult patients with tigecycline-nonsusceptible K. pneumoniae bacteremia in Taiwan using a matched case-control study. We further investigated the tigecycline resistance mechanisms among tigecycline-nonsusceptible K. pneumoniae clinical isolates.

MATERIALS AND METHODS

Study design and population.

This retrospective matched case-control study was conducted at Taipei Veterans General Hospital (a 2,900-bed tertiary-care teaching hospital) in Taipei, Taiwan, from January 2011 through June 2015. The study protocol was approved by the Institutional Review Board at Taipei Veterans General Hospital.

We performed chart reviews for consecutive patients who had blood cultures positive for K. pneumoniae. The cases were patients with tigecycline-nonsusceptible K. pneumoniae bacteremia, and the controls were patients with tigecycline-susceptible K. pneumoniae bacteremia. Nonsusceptibility to tigecycline in a sample was defined by a MIC for tigecycline >2 mg/liter, according to the criteria from the U.S. Food and Drug Administration (15). Controls were matched to cases in a 1:1 ratio based on age, sex, and the at-risk period (the time from admission to the date when the culture was positive). The age and the at-risk period were matched as close as possible. We included the first blood cultures for patients with two or more positive blood cultures. Patients <20 years of age and those with incomplete medical records were excluded.

Data collection.

Medical records were reviewed to obtain clinical information on the demographic characteristics, location at time of culture, the source of bacteremia, comorbidities, immunosuppression, surgeries, invasive procedures or devices, surgical drainage, mechanical ventilation, antimicrobial therapy, severity of illness, outcome, and mortality. Nosocomial infections were defined as K. pneumoniae-positive isolates identified in patients more than 48 h after admission to the hospital. Healthcare-associated infections were defined as patients meeting any of the following criteria: having received intravenous therapy at home or in an outpatient clinic during the previous 30 days, having received renal dialysis in a hospital or clinic during the previous 30 days, having hospitalization for 2 or more days during the previous 90 days, or having resided in a nursing home or long-term-care facility (8). Appropriate empirical antimicrobial therapy was defined as administration of at least one antimicrobial agent to which the causative pathogen was susceptible within 24 h after the onset of bacteremia, at the approved route and dosage for the affected target organ(s). Appropriate definite antimicrobial therapy was defined as administration of at least one antimicrobial agent to which the causative pathogen was susceptible after the report of antimicrobial susceptibility test, at the approved route and dosage for the affected target organ(s). Prior antibiotic exposure was defined as at least 2 days of therapy administered within 30 days prior to the bacteremia. The Pitt bacteremia score and the Acute Physiology and Chronic and Prevention Evaluation (APACHE) II score were calculated to determine the severity of illness within 24 h of the onset of bacteremia (2, 16).

Microbiological studies.

Identification of K. pneumoniae was performed using the Vitek2 system (bioMérieux, Marcy l'Etoile, France) or matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (bioMérieux SA, Marcy l'Etoile, France). Antimicrobial susceptibility on this bacterium was performed using the Vitek2 system (bioMérieux) and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (17). Tigecycline-nonsusceptible K. pneumoniae (tigecycline MIC > 2 mg/liter) in an isolate was further confirmed by the Etest (bioMérieux) method according to the manufacturer's instructions. MDR in K. pneumoniae, as a phenotype, was defined in the isolates as nonsusceptibility to at least one agent in three or more antimicrobial categories (18).

Tigecycline resistance mechanism: overexpression of efflux pumps examined by qRT-PCR and detection of mutations in ramR, acrR, and oqxR and the 30S ribosomal protein S10 encoding region (rpsJ).

All tigecycline-nonsusceptible K. pneumoniae and randomly selected tigecycline-susceptible K. pneumoniae isolates were examined by real-time quantitative reverse transcription-PCR (qRT-PCR) for the mRNA expression level of pump genes (acrA, oqxA, and kpgA), regulators of the acrAB operon (ramA), and transcriptional activator of the efflux pump OqxAB (rarA). The detailed methods were described previously (19). The relative expression of each target gene was then calibrated against the corresponding expression of a tigecycline-susceptible K. pneumoniae KP478, which served as reference strain (expression = 1; tigecycline MIC, 0.25 mg/liter). Specific primers for the aforementioned genes are listed in Table S1 in the supplemental material (2022). The experiments were performed in triplicate. Data analysis used the cycle threshold (ΔΔCT) method, reported values were averaged across all experiments, and the standard errors of the means were calculated.

The presence of mutations in the ramR (23), oqxR (24), and rpsJ (25) were compared to the reference tigecycline-susceptible K. pneumoniae strain MGH78578 (CP000647) (23). The primers were described in Table S1 in the supplemental material.

Statistical analyses.

We used chi-square and Fisher exact test to analyze categorical variables. We used the Student t test and Mann-Whitney rank sum test (Wilcoxon rank-sum test) to analyze continuous variables. To identify independent predictors of tigecycline-nonsusceptible K. pneumoniae bacteremia, we used logistic regression. All biologically possible variables with P values of < 0.1 in univariate analyses were incorporated into a model using a backward approach. The mortality attributable to tigecycline nonsusceptibility was calculated by subtracting the mortality rate of patients with tigecycline-susceptible K. pneumoniae bacteremia from the mortality rate of patients with tigecycline-nonsusceptible K. pneumoniae bacteremia (26).

For all calculations, a two-tailed P value of <0.05 was considered statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software, version 17.0 (SPSS, Inc., Chicago, IL).

RESULTS

During the 54-month study period, 43 cases with tigecycline-nonsusceptible K. pneumoniae bacteremia and 43 matched controls with tigecycline-susceptible K. pneumoniae bacteremia were identified. The risk factors of tigecycline-nonsusceptible K. pneumoniae bacteremia are listed in Table 1. Tigecycline-nonsusceptible K. pneumoniae bacteremia was more likely to be associated with any antibiotic exposure (72.1% versus 41.9%, P = 0.005). Prior use of fluoroquinolone (25.6% versus 2.3%, P = 0.003), third- or fourth-generation cephalosporin (20.9% versus 2.3%, P = 0.015), and metronidazole (18.6% versus 2.3%, P = 0.03) were significantly more common in tigecycline-nonsusceptible K. pneumoniae bacteremia than that in tigecycline-susceptible K. pneumoniae bacteremia. Table 2 showed clinical outcomes of patients in case and control group. Patients in case group were more likely to receive inappropriate empirical antimicrobial therapy than the control group (53.5% versus 9.3%, P < 0.001). The 14-day mortality for tigecycline-nonsusceptible K. pneumoniae was 30.2%, and the 28-day mortality of tigecycline-nonsusceptible K. pneumoniae was 41.9%. The 14- and 28-day mortality attributable to tigecycline nonsusceptibility was 9.3 and 18.6%, respectively.

TABLE 1.

Bivariate analyses of risk factors for tigecycline-nonsusceptible K. pneumoniae bacteremiaa

Variable Tigecycline nonsusceptible (n = 43) Tigecycline susceptible (n = 43) P
Demographics
    Mean age (yrs) ± SD 70.81 ± 13.87 70.79 ± 13.90 0.997
    No. of male patients 30 (69.8) 30 (69.8) 1.000
Median days (IQR) of hospitalization prior to culture 14.0 (1.0–35.0) 7.0 (1.0–16.0) 0.078
Location at time of culture
    Medical ward 19 (44.2) 22 (51.2) 0.517
    Surgical ward 5 (11.6) 7 (16.3) 0.534
    Intensive care unit 8 (18.6) 3 (7.0) 0.195
    Emergency department 11 (25.6) 11 (25.6) 1.000
Nosocomial infection 28 (65.1) 26 (60.5) 0.655
Healthcare-associated infection 9 (20.9) 13 (30.2) 0.323
Community-acquired infection 6 (14.0) 4 (9.3) 0.738
Infection source
    Respiratory tract 12 (27.9) 9 (20.9) 0.451
    Urinary tract 4 (9.3) 3 (7.0) 1.000
    Intra-abdomen 6 (14.0) 10 (23.3) 0.268
    Skin and soft tissue 2 (4.7) 1 (2.3) 1.000
    Intravenous device 1 (2.3) 1 (2.3) 1.000
    Biliary tract 7 (16.3) 5 (11.6) 0.534
    Primary bacteremia 11 (25.6) 14 (32.6) 0.476
Comorbidity
    Malignancy type
        Hematological 8 (18.6) 8 (18.6) 1.000
        Solid 15 (34.9) 20 (46.5) 0.272
    Diabetes mellitus 18 (41.9) 11 (25.6) 0.110
    Chronic kidney disease 18 (41.9) 13 (30.2) 0.261
    Hemodialysis 6 (14.0) 4 (9.3) 0.738
    Congestive heart failure 3 (7.0) 2 (4.7) 1.000
    Liver cirrhosis 3 (7.0) 2 (4.7) 1.000
    Cerebral vascular disease 7 (16.3) 6 (14.0) 0.763
    Dementia 4 (9.3) 3 (7.0) 1.000
    Chronic obstructive lung disease 6 (14.0) 3 (7.0) 0.483
    Collagen vascular disease 1 (2.3) 1 (2.3) 1.000
    Transplantation 3 (7.0) 0 (0.0) 0.241
Immunosuppressionb 15 (34.9) 17 (39.5) 0.655
Surgery within 2 weeks 4 (9.3) 5 (11.6) 1.000
Invasive procedures and devices
    Central venous catheter 13 (30.2) 8 (18.6) 0.209
    Nasogastric/nasojejunal tube 7 (16.3) 10 (23.3) 0.417
    Urinary catheter 15 (34.9) 7 (16.3) 0.048
    Endotracheal tube 2 (4.7) 2 (4.7) 1.000
    Tracheostomy 3 (7.0) 3 (7.0) 1.000
    Surgical drainage 5 (11.6) 4 (9.3) 1.000
Mechanical ventilation 4 (9.3) 2 (4.7) 0.676
Prior antibiotic exposure
    First- or second-generation cephalosporinc 8 (18.6) 5 (11.6) 0.366
    Third- or fourth-generation cephalosporind 9 (20.9) 1 (2.3) 0.015
    β-Lactam and β-lactamase inhibitore 12 (27.9) 10 (23.3) 0.621
    Carbapenemf 7 (16.3) 4 (9.3) 0.520
    Fluoroquinoloneg 11 (25.6) 1 (2.3) 0.003
    Aminoglycosideh 2 (4.7) 1 (2.3) 1.000
    Tigecycline 5 (11.6) 3 (7.0) 0.713
    Glycopeptidei 8 (18.6) 6 (14.0) 0.559
    Metronidazole 8 (18.6) 1 (2.3) 0.030
Median Pitt bacteremia score (IQR) 3.0 (0.0–5.0) 3.0 (2.0–5.0) 0.372
Pitt bacteremia score ≥4 15 (34.9) 15 (34.9) 1.000
Median APACHE II score (IQR) 22.0 (17.0–29.0) 18.0 (13.0–23.0) 0.059
a

Data are presented as the number (%) of patients, unless stated otherwise in column 1. SD, standard deviation; IQR, interquartile range; APACHE, Acute Physiology and Chronic Health Evaluation.

b

Immunosuppression was defined as meeting one of the following criteria: neutropenia, use of corticosteroids, or receiving chemotherapy.

c

Includes cefazolin and cefuroxime.

d

Includes cefoperazone, ceftriaxone, cefotaxime, cefepime, and cefpirome.

e

Includes amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, and ticarcillin-clavulanate.

f

Includes ertapenem, imipenem, meropenem, and doripenem.

g

Includes ciprofloxacin, levofloxacin, and moxifloxacin.

h

Includes amikacin, gentamicin, and isepamicin.

i

Includes vancomycin and teicoplanin.

TABLE 2.

Clinical outcomes of patients with K. pneumoniae bacteremiaa

Variable Tigecycline nonsusceptible (n = 43) Tigecycline susceptible (n = 43) P
Appropriate empirical antimicrobial therapy 20 (46.5) 39 (90.7) <0.001
Appropriate definite antimicrobial therapy 35 (81.4) 39 (90.7) 0.351
Median length of stay in days after bacteremia (IQR) 13.0 (8.0–25.0) 14.0 (6.0–23.0) 0.983
Septic shock when bacteremia 23 (53.5) 31 (72.1) 0.074
Mortality
    In-hospital mortality 18 (41.9) 12 (27.9) 0.175
    14-day mortality 13 (30.2) 9 (20.9) 0.323
    28-day mortality 18 (41.9) 10 (23.3) 0.066
a

Data are presented as the number (%) of patients, unless stated otherwise in column 1. IQR, interquartile range.

The antimicrobial susceptibilities of the K. pneumoniae clinical isolates from this study showed that tigecycline-nonsusceptible K. pneumoniae exhibited a higher rate of MDRKP than the tigecycline-susceptible ones (88.4% versus 30.2%, P < 0.001). The resistant rates for gentamicin, ceftriaxone, ceftazidime, cefepime, ciprofloxacin, and levofloxacin were statistically higher in tigecycline-nonsusceptible K. pneumoniae isolates than those of the tigecycline-susceptible K. pneumoniae ones (Table 3). We also found less than one-fifth of isolates were resistant to carbapenem.

TABLE 3.

Antimicrobial susceptibilities of clinical isolates of K. pneumoniaea

Antimicrobial agent Tigecycline nonsusceptible (n = 43) Tigecycline susceptible (n = 43) P
Amikacin 3 (7.0) 2 (4.7) 1.000
Gentamicin 19 (44.2) 8 (18.6) 0.011
Ceftriaxone 21 (48.8) 9 (20.9) 0.007
Ceftazidime 24 (55.8) 10 (23.3) 0.002
Cefepime 18 (41.9) 7 (16.3) 0.009
Ciprofloxacin 30 (69.6) 7 (16.3) <0.001
Levofloxacin 30 (69.6) 7 (16.3) <0.001
Ertapenem 8 (18.6) 5 (11.6) 0.366
Imipenem 2 (4.7) 4 (9.3) 0.397
a

Data are presented as the number (%) of isolates resistant to the antibiotic indicated.

All plausible biological risk factors for tigecycline-nonsusceptible K. pneumoniae bacteremia analyzed in the univariate analyses with P values of <0.1 were further analyzed in the multivariate analyses. Fluoroquinolone use within 30 days prior to bacteremia (odds ratio, 10.81; 95% confidence interval, 1.26 to 93.13; P = 0.030) was the only independent risk factor for tigecycline-nonsusceptible K. pneumoniae bacteremia (Table 4).

TABLE 4.

Multivariate analyses of risk factors for tigecycline-nonsusceptible K. pneumoniae bacteremia

Variable OR (95% CI)a P
Urinary catheter 2.29 (0.72–7.32) 0.163
Prior exposure to third- or fourth-generation cephalosporinsb 7.36 (0.81–67.14) 0.077
Prior exposure to fluoroquinolonec 10.81 (1.26–93.13) 0.030
Prior exposure to metronidazole 5.60 (0.58–53.9) 0.136
Septic shock when bacteremia 0.47 (0.17–1.28) 0.139
a

OR, odds ratio; CI, confidence interval.

b

Includes cefoperazone, ceftriaxone, cefotaxime, cefepime, and cefpirome.

c

Includes ciprofloxacin, levofloxacin, and moxifloxacin.

Table S2 in the supplemental material detailed the resistance mechanisms of 43 tigecycline-nonsusceptible K. pneumoniae isolates. Twenty-three (TNSKP1 to -23) isolates had elevated expression levels of acrA (>2-fold) but not of rarA and oqxA (<4-fold). Most of them (TNSKP5 to -22) had elevated expression levels of both ramA and acrA (>2-fold), but 4 isolates (TNSKP1 to -4) had a baseline expression level of ramA (<2-fold). Five isolates (TNSKP24 to -28) had elevated expression levels of oqxA (>4-fold), but not of ramA and acrA (<2-fold). Thirteen isolates (TNSKP29 to -41) had higher expression levels of both acrA and oqxA. To verify the role of efflux pumps in tigecycline resistance, we randomly selected 7 tigecycline-susceptible K. pneumoniae isolates from the control group for the same experiments, and only one of them (tigecycline MIC = 1 mg/liter) showed elevated expression level of both acrA and oqxA compared to the reference KP478 strain. These data indicate that tigecycline nonsusceptibility in most of these isolates was caused by the overexpression of the AcrAB and/or OqxAB efflux pumps. The upregulation of RamA and/or RarA contributed to the majorities of efflux pumps overexpression. Interestingly, we found 16 isolates had overexpression of kpgA (>100-fold), in addition to the overexpression of the AcrAB and/or OqxAB efflux pumps. One isolate (TNSKP42, MIC = 4 mg/liter) showed overexpression of kpgA but baseline expression of these 4 efflux-related genes. Finally, we found the remaining 1 TNSKP isolate (TNSKP43, MIC = 3 mg/liter) exhibited baseline expression of these 5 efflux-related genes. No mutation in rpsJ, coding for ribosomal protein S10, was detected in all the 43 isolates.

DISCUSSION

This study investigated the risk factors for tigecycline-nonsusceptible K. pneumoniae bacteremia using a matched case-control study, and elucidated possible mechanisms for tigecycline nonsusceptibility among tigecycline-nonsusceptible K. pneumoniae isolates. We found that fluoroquinolone use in the previous 30 days was the only independent risk factor for tigecycline-nonsusceptible K. pneumoniae bacteremia. Tigecycline-nonsusceptible K. pneumoniae bacteremia was associated with high 14- and 28-day mortality in the patients. The 14- and 28-day mortality attributable to tigecycline nonsusceptibility was 9.3 and 18.6%, respectively.

Despite the growing clinical concerns about the increasing prevalence of tigecycline-nonsusceptible K. pneumoniae isolates, the impact and clinical characteristics of patients with tigecycline-nonsusceptible K. pneumoniae infections are rarely reported in the scientific literature (8). In our previous study, we reported the high 28-day mortality among patients with tigecycline-nonsusceptible K. pneumoniae bacteremia (8). Here, we further compared tigecycline-nonsusceptible K. pneumoniae and tigecycline-susceptible K. pneumoniae bacteremia and demonstrated the mortality attributable to tigecycline nonsusceptibility. Given the high mortality of tigecycline-nonsusceptible K. pneumoniae bacteremia, better control measures and efficient surveillance are necessary.

Nigo et al. performed a nested case-control study on the emergence of tigecycline-resistant MDRKP isolates among patients who initially presented with a tigecycline-susceptible MDRKP isolate (10). They found that receipt of tigecycline was the only independent predictor of subsequent isolation of a tigecycline-resistant strain in the study (10). van Duin et al. described that tigecycline use was independently associated with the development of subsequent tigecycline resistance in the same patient with CRKP bacteriuria (6). In the present study, we used a different study design to those of Nigo et al. and van Duin et al. (6, 10). The previous two studies focused on patients who initially presented with tigecycline-nonsusceptible K. pneumoniae in their urine (6) or other sites (10), regardless of colonization or infection. Our inclusion of patients with K. pneumoniae bacteremia was not limited to those who initially presented with tigecycline-susceptible K. pneumoniae isolates. We found that most tigecycline-nonsusceptible K. pneumoniae isolates from blood were susceptible to carbapenems, which was consistent to our previous study (8). The present study showed that only 5 cases had prior use of tigecycline. One study from India also showed that tigecycline nonsusceptibility was not observed among CRKP isolates, and the increase of MIC values of tigecycline was not associated with the use of tigecycline (27). The geographic differences might be responsible for the different resistance phenotype of tigecycline-nonsusceptible K. pneumoniae between Asia and western countries.

The present study showed that prior fluoroquinolone exposure was the only independent risk factor for tigecycline-nonsusceptible K. pneumoniae bacteremia. Overexpression of the AcrAB multidrug efflux system has been reported to contribute to fluoroquinolone resistance in Klebsiella spp. (28). Cross-resistance to olaquindox, chloramphenicol, and the quinolones due to elevated expression of the chromosomal oqxAB operon in K. pneumoniae has been reported recently (29). Recent studies have established that overexpression of AcrAB efflux pump plays a major role in tigecycline resistance in K. pneumoniae (23, 30). Overexpression of OqxAB has contributed to tigecycline resistance in one study (12). The previous use of fluoroquinolones that are also effluxed through the AcrAB or OqxAB pump might result in overexpression of these efflux pumps and subsequently reduce susceptibility to tigecycline. In one recent study, Roy et al. reported that the increment of MIC values of tigecycline in K. pneumoniae might be related to the use of other antibiotics that are also the substrates the AcrAB pump because of the restricted use of tigecycline in their unit (27). Furthermore, our recent study regarding in vivo evolution of tigecycline resistance in patients demonstrated the cases infected with tigecycline-nonsusceptible strains derived from their isogenic tigecycline-susceptible ones. We found two cases had been exposed to fluoroquinolones before the appearance of the tigecycline-nonsusceptible strains (19). The results from the above-mentioned studies might support our important finding that tigecycline-nonsusceptible K. pneumoniae bacteremia can occur among patients recently treated with fluoroquinolones. The present study provides insight on the issue about antibiotic exposure and resistance.

Regarding mechanisms for tigecycline resistance, most studies have emphasized that the efflux pump AcrAB, which can be upregulated by ramR mutations and subsequent ramA activation, contributed to reduced susceptibility to tigecycline in K. pneumoniae clinical isolates (1214, 24, 31). However, the role of OqxAB pump in tigecycline resistance was either undefined (14, 24) or not described (31) in some studies. In the present study, most tigecycline-nonsusceptible K. pneumoniae isolates had elevated expression levels of ramA and acrA. Several tigecycline-nonsusceptible K. pneumoniae isolates had markedly elevated expression levels of rarA and oqxA, but baseline expression level of acrA. The results presented here support a previous report that rarA is one of the regulator pathways that controls the expression of oqxA in K. pneumoniae and contributes to tigecycline nonsusceptibility (21). OqxR has been suggested as a repressor not only of the oqxAB efflux operon, but also of rarA (24). Interestingly, we observed higher expression of oqxA and rarA in several clinical isolates with a silence mutation of oqxR (see Table S2 in the supplemental material). These isolates could harbor additional genetic alterations responsible for the overexpression of oqxA or rarA. Among tigecycline-susceptible K. pneumoniae isolates, we only found one isolate had higher expression of acrA and oqxA, which further supported the role of the above-mentioned efflux pumps in tigecycline resistance.

Interestingly, we found one strain (TNSKP42) with the isolated overexpression of kpgA leading to tigecycline nonsusceptibility. Our findings might suggest kpgABC play some role in tigecycline resistance. Finally, the reported regulatory pathways of tigecycline resistance were completely absent in one tigecycline-nonsusceptible K. pneumoniae isolate, which indicated that alternative regulatory pathways may exist in tigecycline resistance mechanisms.

Our study was limited by several factors. The case numbers in this study were relatively small. Although there were limited numbers of tigecycline-nonsusceptible K. pneumoniae bacteremia clinically, the present study still represents the largest collection in the literature. Being retrospective in nature, there might be selection bias in this study. The matched case-control study design helped limit this type of bias. Another limitation of this study is that data on prior antibiotic usage were evaluated only for the 1-month period prior to the bacteremia. This may have resulted in an underestimation of antibiotic usage. Finally, because the case and control groups were from a single medical center, it is possible that the results are not generalizable.

In conclusion, patients with tigecycline-nonsusceptible K. pneumoniae bacteremia were associated with high mortality. Our matched case-control study has shown that fluoroquinolone use within 30 days prior to bacteremia in patients was the only independent risk factor for tigecycline-nonsusceptible K. pneumoniae bacteremia. This study further underscores the role of AcrAB and OqxAB in tigecycline-nonsusceptible K. pneumoniae.

Supplementary Material

Supplemental material

ACKNOWLEDGMENTS

We thank Chiu-Mei Yeh for her support and assistance in the statistical analyses. We also thank the Medical Science and Technology Building of Taipei Veterans General Hospital for providing experimental space and facilities.

This study was partly supported by grants from the Ministry of Science and Technology in Taiwan (MOST 105-2628-B-010-015-MY3), the Taipei Veterans General Hospital (V103B-016, V104B-001, and V105B-001), and the Szu-Yuan Research Foundation of Internal Medicine (grant 105001).

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01503-16.

REFERENCES

  • 1.Meatherall BL, Gregson D, Ross T, Pitout JD, Laupland KB. 2009. Incidence, risk factors, and outcomes of Klebsiella pneumoniae. Am J Med 122:866–873. doi: 10.1016/j.amjmed.2009.03.034. [DOI] [PubMed] [Google Scholar]
  • 2.Paterson DL, Ko WC, Von Gottberg A, Mohapatra S, Casellas JM, Goossens H, Mulazimoglu L, Trenholme G, Klugman KP, Bonomo RA, Rice LB, Wagener MM, McCormack JG, Yu VL. 2004. International prospective study of Klebsiella pneumoniae: implications of extended-spectrum beta-lactamase production in nosocomial infections. Ann Intern Med 140:26–32. doi: 10.7326/0003-4819-140-1-200401060-00008. [DOI] [PubMed] [Google Scholar]
  • 3.Braykov NP, Eber MR, Klein EY, Morgan DJ, Laxminarayan R. 2013. Trends in resistance to carbapenems and third-generation cephalosporins among clinical isolates of Klebsiella pneumoniae in the United States, 1999-2010. Infect Control Hosp Epidemiol 34:259–268. doi: 10.1086/669523. [DOI] [PubMed] [Google Scholar]
  • 4.Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. 2012. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev 25:682–707. doi: 10.1128/CMR.05035-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Pournaras S, Koumaki V, Spanakis N, Gennimata V, Tsakris A. 2016. Current perspectives on tigecycline resistance in Enterobacteriaceae: susceptibility testing issues and mechanisms of resistance. Int J Antimicrob Agents 48:11–18. doi: 10.1016/j.ijantimicag.2016.04.017. [DOI] [PubMed] [Google Scholar]
  • 6.vain Duin D, Cober ED, Richter SS, Perez F, Cline M, Kaye KS, Kalayjian RC, Salata RA, Evans SR, Fowler VG Jr, Bonomo RA. 2014. Tigecycline therapy for carbapenem-resistant Klebsiella pneumoniae (CRKP) bacteriuria leads to tigecycline resistance. Clin Microbiol Infect 20:O1117–O1120. doi: 10.1111/1469-0691.12714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Arya SC, Agarwal N. 2010. Emergence of tigecycline resistance amongst multi-drug resistant gram negative isolates in a multi-disciplinary hospital. J Infect 61:358–359. doi: 10.1016/j.jinf.2010.07.012. [DOI] [PubMed] [Google Scholar]
  • 8.Lin YT, Wang FD, Chan YJ, Fu YC, Fung CP. 2014. Clinical and microbiological characteristics of tigecycline non-susceptible Klebsiella pneumoniae in Taiwan. BMC Infect Dis 14:1. doi: 10.1186/1471-2334-14-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Sader HS, Flamm RK, Jones RN. 2013. Tigecycline activity tested against antimicrobial resistant surveillance subsets of clinical bacteria collected worldwide (2011). Diagn Microbiol Infect Dis 76:217–221. doi: 10.1016/j.diagmicrobio.2013.02.009. [DOI] [PubMed] [Google Scholar]
  • 10.Nigo M, Cevallos CS, Woods K, Flores VM, Francis G, Perlman DC, Revuelta M, Mildvan D, Waldron M, Gomez T, Koshy S, Jodlowski T, Riley W, Ruhe JJ. 2013. Nested case-control study of the emergence of tigecycline resistance in multidrug-resistant Klebsiella pneumoniae. Antimicrob Agents Chemother 57:5743–5746. doi: 10.1128/AAC.00827-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sun Y, Cai Y, Liu X, Bai N, Liang B, Wang R. 2013. The emergence of clinical resistance to tigecycline. Int J Antimicrob Agents 41:110–116. doi: 10.1016/j.ijantimicag.2012.09.005. [DOI] [PubMed] [Google Scholar]
  • 12.Sheng ZK, Hu F, Wang W, Guo Q, Chen Z, Xu X, Zhu D, Wang M. 2014. Mechanisms of tigecycline resistance among Klebsiella pneumoniae clinical isolates. Antimicrob Agents Chemother 58:6982–6985. doi: 10.1128/AAC.03808-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhong X, Xu H, Chen D, Zhou H, Hu X, Cheng G. 2014. First emergence of acrAB and oqxAB mediated tigecycline resistance in clinical isolates of Klebsiella pneumoniae pre-dating the use of tigecycline in a Chinese hospital. PLoS One 9:e115185. doi: 10.1371/journal.pone.0115185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.He F, Fu Y, Chen Q, Ruan Z, Hua X, Zhou H, Yu Y. 2015. Tigecycline susceptibility and the role of efflux pumps in tigecycline resistance in KPC-producing Klebsiella pneumoniae. PLoS One 10:e0119064. doi: 10.1371/journal.pone.0119064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.U.S. Food and Drug Administration. Tygacil (tigecycline). 2009. U.S. Food and Drug Administration, Washington, DC: http://www.fda.gov/Safety/MedWatch/SafetyInformation/Safety-RelatedDrugLabelingChanges/ucm132714.htm. [Google Scholar]
  • 16.Knaus WA, Draper EA, Wagner DP, Zimmerman JE. 1985. APACHE II: a severity of disease classification system. Crit Care Med 13:818–829. doi: 10.1097/00003246-198510000-00009. [DOI] [PubMed] [Google Scholar]
  • 17.Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial susceptibility testing; 26th informational supplement. Document M100-S26. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
  • 18.Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268–281. doi: 10.1111/j.1469-0691.2011.03570.x. [DOI] [PubMed] [Google Scholar]
  • 19.Lin YT, Huang YW, Huang HH, Yang TC, Wang FD, Fung CP. 2016. In vivo evolution of tigecycline non-susceptible Klebsiella pneumoniae strains in patients: relationship between virulence and resistance. Int J Antimicrob Agents 2016:S0924-8579(16)30204-7. doi: 10.1016/j.ijantimicag.2016.07.008. [DOI] [PubMed] [Google Scholar]
  • 20.Källman O, Motakefi A, Wretlind B, Kalin M, Olsson-Liljequist B, Giske CG. 2008. Cefuroxime non-susceptibility in multidrug-resistant Klebsiella pneumoniae overexpressing ramA and acrA and expressing ompK35 at reduced levels. J Antimicrob Chemother 62:986–990. doi: 10.1093/jac/dkn296. [DOI] [PubMed] [Google Scholar]
  • 21.Veleba M, Higgins PG, Gonzalez G, Seifert H, Schneiders T. 2012. Characterization of RarA, a novel AraC family multidrug resistance regulator in Klebsiella pneumoniae. Antimicrob Agents Chemother 56:4450–4458. doi: 10.1128/AAC.00456-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Nielsen LE, Snesrud EC, Onmus-Leone F, Kwak YI, Avilés R, Steele ED, Sutter DE, Waterman PE, Leshoa EP. 2014. IS5 element integration, a novel mechanism for rapid in vivo emergence of tigecycline nonsusceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 58:6151–6156. doi: 10.1128/AAC.03053-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Hentschke M, Wolters M, Sobottka I, Rohde H, Aepfelbacher M. 2010. ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to tigecycline. Antimicrob Agents Chemother 54:2720–2723. doi: 10.1128/AAC.00085-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Bialek-Davenet S, Lavigne JP, Guyot K, Mayer N, Tournebize R, Brisse S, Leflon-Guibout V, Nicolas-Chanoine MH. 2015. Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae. J Antimicrob Chemother 70:81–88. doi: 10.1093/jac/dku340. [DOI] [PubMed] [Google Scholar]
  • 25.Villa L, Feudi C, Fortini D, García-Fernández A, Carattoli A. 2014. Genomics of KPC-producing Klebsiella pneumoniae sequence type 512 clone highlights the role of RamR and ribosomal S10 protein mutations in conferring tigecycline resistance. Antimicrob Agents Chemother 58:1707–1712. doi: 10.1128/AAC.01803-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Falagas ME, Tansarli GS, Karageorgopoulos DE, Vardakas KZ. 2014. Deaths attributable to carbapenem-resistant Enterobacteriaceae infections. Emerg Infect Dis 20:1170–1175. doi: 10.3201/eid2007.121004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Roy S, Datta S, Viswanathan R, Singh AK, Basu S. 2013. Tigecycline susceptibility in Klebsiella pneumoniae and Escherichia coli causing neonatal septicaemia (2007-10) and role of an efflux pump in tigecycline non-susceptibility. J Antimicrob Chemother 68:1036–1042. doi: 10.1093/jac/dks535. [DOI] [PubMed] [Google Scholar]
  • 28.Mazzariol A, Zuliani J, Cornaglia G, Rossolini GM, Fontana R. 2002. AcrAB efflux system: expression and contribution to fluoroquinolone resistance in Klebsiella spp. Antimicrob Agents Chemother 46:3984–3986. doi: 10.1128/AAC.46.12.3984-3986.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wong MH, Chan EW, Chen S. 2015. Evolution and dissemination of OqxAB-like efflux pumps, an emerging quinolone resistance determinant among members of Enterobacteriaceae. Antimicrob Agents Chemother 59:3290–3297. doi: 10.1128/AAC.00310-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ruzin A, Visalli MA, Keeney D, Bradford PA. 2005. Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 49:1017–1022. doi: 10.1128/AAC.49.3.1017-1022.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wang X, Chen H, Zhang Y, Wang Q, Zhao C, Li H, He W, Zhang F, Wang Z, Li S, Wang H. 2015. Genetic characterisation of clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline: Role of the global regulator RamA and its local repressor RamR. Int J Antimicrob Agents 45:635–640. doi: 10.1016/j.ijantimicag.2014.12.022. [DOI] [PubMed] [Google Scholar]

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