Summary

Introduction: This study aims to assess the in vitro activity of ceftazidime-avibactam (CZA) and ceftolozane-tazobactam (C/T) against the multi-drug resistant (MDR) Klebsiella pneumoniae. Furthermore, we aimed to determine the types of carbapenemase enzyme responsible for carbapenem resistance and compare the activity of ceftolozane-tazobactam and CZA according to the types of carbapenemase enzymes produced.
Materials and Methods: Twenty-two MDR isolates were investigated in the study. The identification and antimicrobial susceptibilities of the isolates were performed by VITEK 2 (BioMérieux, France) automated system. The activity of C/T and CZA was determined by the gradient strip test (Liofilchem MIC strip test, Italy). In all K. pneumoniae isolates, bla_IMP-1, bla_KPC, bla_NDM-1, bla_OXA-48and bla_VIM gene regions encoding the carbapenemase enzyme were investigated by using Xpert CARBA-R test kits (Cepheid, Sunnyvale, CA, USA) of the Gene-Xpert^® system.
Results: Ceftolozane-tazobactam resistance was detected in 21 (95%) isolates, whereas CZA resistance was detected in six (27%) isolates. Among the 22 K. pneumoniae isolates, NDM-1 gene region was detected in three, NDM-1+OXA-48 gene region was detected in two, OXA-48 was detected in eight, and KPC gene region was detected in nine isolates. VIM and IMP-1 gene regions were not detected. Ceftolozane-tazobactam resistance was detected in seven isolates, whereas CZA resistance was not detected among the eight K. pneumoniae isolates producing only OXA-48. NDM-1 and OXA-48 co-producing isolates were detected resistant to C/T and CZA with high minimum inhibitory concentration (MIC) levels (MIC ≥256 µg/ml). All NDM-1 producing isolates had high MIC levels (MIC ≥256 µg/ml) to both C/T and CZA. Ceftazidime-avibactam resistance was detected in only one isolate, whereas C/T resistance was detected in all K. pneumoniae isolates producing KPC. Ceftolozane-tazobactam was detected inefficient whereas CZA was found very efficient in MDR K. pneumoniae isolates producing KPC.
Conclusion: According to the obtained data, we detected the in vitro antibacterial activity of CZA against the MDR K. pneumoniae isolates was superior to that of C/T. Ceftolozane-tazobactam was found to be weakly efficient, whereas CZA was found to be highly efficient against the MDR K. pneumoniae isolates producing OXA-48 and KPC. Because avibactam can inhibit the activity of KPC-type carbapenemase, the combination drug CZA should be considered to be effective in the treatment of KPC-type carbapenemase-producing strains but noneffective against NDM-type carbapenemase-producing strains.

Introduction

Antibiotic resistance encountered in Gram-negative bacteria, especially the Enterobacteriaceae family and non-fermentative bacteria, has led to a decrease in antibiotic options used in the treatment of infections caused by these bacteria, thus increasing the morbidity and mortality rates^{[1, 2]}. The production of extended spectrum beta-lactamase (ESBL) is common in Gram-negative bacteria. In recent years, the production of carbapenemase has also increased, leading to multi-drug resistance (MDR)^[3].

In the last 20 years, the number of newly developed and used antibiotics has been decreasing. For this reason, some antibiotics are used in combination nowadays to benefit from their synergistic effects. Ceftolozane, a new third generation cephalosporin, has been put into use in combination with the beta-lactamase inhibitor tazobactam. This antibiotic has been found effective against most Gram-negative bacteria, including the Enterobacteriaceae family. Ceftolozane-tazobactam (C/T) (Zerbaxa; Merck & Co., Kenilworth, NJ, USA) was approved for use in complicated intraabdominal infections and complicated urinary tract infections including pyelonephritis in 2014^{[1, 4]}. Ceftazidime-avibactam (CZA) (Avycaz; Allergen, Inc., Irvine, CA, USA) is another third-generation cephalosporin and beta-lactamase inhibitor combination that was approved for use in the same infections as C/T in 2015^{[1, 4]}. Ceftazidime-avibactam has the same spectrum of activity as ceftazidime against Enterobacteriaceae. However, the addition of avibactam extended its activity against resistant strains carrying certain beta-lactamases^[1]. Ceftolozane, a new oxyimino-aminothiazolyl cephalosporin with a structure similar to ceftazidime, is less sensitive to hydrolysis with AmpC in contrast to ceftazidime and is also less affected by porin loss than ceftazidime. Tazobactam and avibactam inhibit serine beta lactamase. In addition to inhibiting ESBL, avibactam also effectively inhibits Class A carbapenemases such as AmpC beta-lactamases and KPC^{[5, 6]}. However, it has no activity against metallo beta-lactamases^[3].

There are few studies investigating the effectiveness of CZA and C/T in MDR Klebsiella pneumoniae isolates and comparing their efficacy in these bacteria^{[7, 8]}. In our study, it was aimed to investigate the in vitro efficiency of CZA and C/T in MDR Klebsiella pneumoniae isolates. In addition, it was aimed to determine the types of carbapenemase enzymes that cause carbapenem resistance and to compare the efficiency of CZA and C/T according to these enzymes.

Methods

Bacterial Isolates and Antibiotic Susceptibility Tests

Approval for this study was obtained from Sakarya University Faculty of Medicine Ethics Committee (ethics committee approval no: 71522473/050.0104/75, date: 16.08.2017). Within the scope of this study, identification and antibiotic susceptibility tests of 22 MDR K. pneumoniae isolates isolated from various clinical specimens were performed in our laboratory. Susceptibilities of amikacin, gentamicin, ciprofloxacin, levofloxacin, cefepime, ceftriaxone, ceftazidime, piperacillin-tazobactam, cefuroxime-axetil, amoxicillin-clavulanate, imipenem, meropenem and trimethoprim-sulfamethoxazole were studied with VITEK 2 (BioMérieux, Fransa) automated system.

In K. pneumoniae isolates included in the study; strains found resistant to three or more of antimicrobial agents including piperacillin-tazobactam, cefepime, meropenem, ciprofloxacin, and aminoglycosides, were considered MDR^[9].

Minimum Inhibitor Concentration (MIC) Tests

The C/T and CZA activities were determined by the gradient strip test (Liofilchem MIC strip test, Italy). Antimicrobial susceptibility results were evaluated according to the CLSI 2018 criteria^[10]. For CZA; MIC ≤8 µg/ml is considered susceptible and ≥16 µg/ml is considered resistant. For C/T; MIC ≤2 µg/ml is considered susceptible, MIC=4 µg/ml is considered medium sensitive, and MIC ≥8 µg/ml is considered resistant^[11].

Carbapenem resistance was confirmed by restudying gradient test strips (E-test, BioMérieux, France). Isolates that were ertapenem resistant (≥2 µg/ml) and/or meropenem resistant (≥4 µg/ml) were defined as carbapenem resistant according to MIC values.

Modified Carbapenemase Inactivation Test (CIT)

A loopful was taken from the isolate in which carbapenemase enzyme production would be investigated, suspended in sterile distilled water and 10 µl Meropenem disc (BioMérieux, France) was thrown into it. After two hours of incubation, the Meropenem disc from the suspension was placed on E. coli ATCC 29522 inoculated Mueller-Hinton (Merck, USA) agar medium and incubated at 35 °C for six hours. If growth occured around the meropenem disc (no inhibition zone) at the end of the six-hour period, the result of the test was considered positive^[10].

Carbapenemase Nordmann-Poirel (Carba NP) test

The Carba NP (BioMérieux, France) test was studied in accordance with the manufacturer’s recommendations. The bacterial colony to be tested was suspended in lysis buffer and incubated for 30 minutes. This bacterial suspension was centrifuged at 10,000 x g for five minutes at room temperature. The solution prepared using the supernatant, imipenem monohydrate (Sigma, Saint-Quentin Fallavier, France), phenol red and 0.1 mmol/l ZnSO₄ (Merck Millipore, Guyancourt, France) were mixed in 96-well plates. The phenol red solution mixture and the enzymatic suspension were incubated at 37 °C for two hours. The test results were then interpreted.

Molecular Tests

The bla_IMP-1, bla_KPC, bla_NDM-1, bla_OXA-48 and bla_VIMgene regions were investigated with Xpert CARBA-R Test kits (Cepheid, Sunnyvale, CA, USA) of the Gene-Xpert^® system in all Klebsiella isolates included in the study.

After the incubation of carbapenem-resistant isolates in blood agar medium at 35-37 °C for 24 hours, the colonies from fresh colonies of these isolates were transferred to Xpert sample buffer with the help of a sterile swap. Two ml of this suspension was transferred to a commercial ready-to-use cartridge where DNA extraction, amplification and detection reactions would be performed. The cartridge was then loaded into the Gene-Xpert^® instrument and the results were evaluated according to the manufacturer’s instructions within one hour.

Results

All of the MDR K. pneumoniae isolates included in the study were isolated from intensive care units. Twelve (55%) of the clinical samples were included in the study as rectal swab, six (27%) as blood sample, two (9%) as wound sample and two (9%) as urine samples. One of the isolates obtained from more than one clinical sample of the same patient was included in the study. All isolates were evaluated as positive for carbapenemase in the CIT and Carba NP tests which were carried out to detect Carbapenemase production. The clinical, molecular and antibiotic susceptibility test results of the analyzed MDR K. pneumoniae isolates are presented in Table 1.

Table 1: Clinical, molecular and antibiotic susceptibility test results of multi-drug resistant K. pneumoniae isolates

It was determined that the 45% of MDR K. pneumoniae isolates included in the study were resistant to amikacin and 90% were resistant to gentamicin. Ceftazidime-avibactam resistance was observed in six (27%) isolates, while C/T resistance was detected in 21 (95%) isolates. All isolates resistant to CZA were also found to be resistant to C/T.

While 12 (55%) of the C/T-resistant MDR K. pneumoniae isolates were isolated from rectal swab, six (27%) from blood sample, two (9%) from wound sample and one (5%) from urine sample; three (14%) of CZA-resistant K. pneumoniae isolates were isolated from blood sample, two (9%) from rectal swab, and one (5%) from urine sample.

In our study, five gene regions encoding carbapenemase were investigated in 22 carbapenem resistant K. pneumoniae isolates by molecular method. As a result of this investigation, three of these isolates had NDM-1, two had NDM-1/OXA-48, eight had OXA-48 and nine had KPC gene regions. VIM and IMP-1 gene regions were not detected in any isolate.

Among the eight K. pneumoniae isolates producing only OXA-48, seven isolates were resistant to C/T (MIC: 12-256 µg/ml), while all isolates were susceptible to CZA (MIC: 0.5-1.5 µg/ml). A high level of resistance to C/T and CZA (MIC >=256 µg/ml) was detected in isolates with OXA-48 as well as NDM-1 production. While CZA was found to be very effective in MDR K. pneumoniae isolates producing OXA-48, C/T was found to be less effective in these isolates.

A high level of resistance (MIC >256 µg/ml) was detected both against C/T and CZA in all isolates with NDM-1 production. While all KPC-producing K. pneumoniae isolates were found resistant to C/T (MIC: 32-256 µg/ml), resistance to CZA was detected in only one isolate (MIC: 16 µg/ml). While CZA was very effective (MIC: 0.20-3 µg/ml) in MDR K. pneumoniae isolates producing KPC, it was determined that C/T was not effective in these isolates.

Discussion

Today, treatment options remain limited in infections caused by resistant Gram-negative bacteria. However, there are various treatment algorithms recommended according to the source of infection in the treatment of carbapenem-resistant Enterobacteriaceae. It has been reported that combination therapy can reduce mortality in carbapenem-resistant Enterobacteriaceae infections compared with monotherapy^[12]. Options such as aminoglycoside, tigecycline, fosfomycin, and rifampin can be added in addition to meropenem or doripenem as combined treatment options^[15].

The benefits of combination therapy include prevention of inappropriate antibiotic use, potential synergistic effects, and suppression of resistance development^[14]. Since all monotherapy options have significant limitations (pharmacokinetics, toxicity, resistance development), optimization of therapy with combined antibiotics may be an important option. However, with combination therapy, an increased risk of Clostridium difficile infection, the likelihood of colonization or infection with other resistant bacteria or side effects such as nephrotoxicity may be observed^{[14, 16]}.

It has been reported in various publications that the efficiency of C/T and CZA in infections caused by resistant isolates is higher than other cephalosporins and beta lactamase inhibitors^{[17, 18]}. In studies conducted with carbapenem-resistant Enterobacteriaceae isolates, C/T susceptibility was detected between 10-97.3% and CZA susceptibility between 45% and 100%^{[8, 19-29]}. The C/T and CZA resistance rates we examined in our study were determined as 95.4% and 27.2%, respectively, in MDR K. pneumoniae isolates. In addition, CZA and C/T were found to be the most effective drugs in the beta-lactam group, and were found more effective than most antibiotics, including carbapenems and fluoroquinolones.

To date, few studies have been conducted comparing the efficiency of C/T and CZA in resistant Gram-negative bacteria^{[7, 8]}. In a study conducted with carbapenem-resistant Enterobacteriaceae isolates, CZA was found to be more effective than C/T (susceptibility 45% and 10%, respectively)^[8]. The reason for this was shown to be the change in susceptibility according to different carbapenemase types they detected. In various studies performed with carbapenemase-producing Enterobacteriaceae isolates, it has been reported that CZA and C/T susceptibilities may vary according to the carbapenemase enzyme type^{[5, 7, 8]}. In the study of Evans et al.^[30], CZA and C/T susceptibilities were reported as 100% in carbapenem susceptible strains. However, in the same study, CZA and C/T susceptibility rates were reported as 0% in carbapenem-resistant strains producing VIM, 50% in KPC-producing strains, and 73.1% in all strains for C/T and 77.2% in all strains for CZA. In the study of Alatoom et al.^[8], isolates were divided into three groups according to carbapenemase enzyme types. While CZA showed high activity (80%) in the group with OXA-48 positive isolates, it was found to show low activity in isolates in the other two groups containing NDM and NDM+OXA (29% and 5%, respectively). On the other hand, C/T was evaluated as weakly effective (13%, 21% and 0%, respectively) in all three groups. They showed the high number of NDM and NDM+OXA positive isolates (55%) in their study as the reason for the lower CZA susceptibility rates compared to other studies (Table 2).

Table 2: The ceftolozane-tazobactam and ceftazidime-avibactam susceptibility rates in studies with resistant Enterobacteriaceae isolates

In our study, the efficiency of CZA in MDR K. pneumoniae isolates was found to be higher than C/T (27.2% vs. 95.4% resistance, respectively). We thought that this change in susceptibility rates was due to the fact that all isolates in our study were MDR and the production of carbapenemase we detected in all of them. In addition, variation in carbapenemase-producing genes or differences in resistance mechanisms to both drugs may also have affected this result^[31]. Ceftazidime-avibactam has also been reported to be effective in the treatment of KPC-type carbapemenase-producing isolates, especially since avibactam can inhibit KPC type carbapemenase activity^[8]. In our study, in accordance with the literature, while CZA was found to be very effective in MDR K. pneumoniae isolates producing KPC, it was determined that C/T was not effective in these isolates. Similarly, while CZA was found to be very effective in MDR K. pneumoniae isolates producing OXA-48, it was determined that the efficiency of C/T in these isolates was low. In the NDM-1 producing MDR K. pneumoniae isolates, a high value of resistance (MIC >=256 µg/ml) was found against both C/T and CZA.

The fact that C/T and CZA activities can change according to carbapenemase production reveals the importance of detecting carbapenemase resistance genes. In the study of Wang et al.^[32], CZA MIC₉₀ values in E. cloacae and K. pneumoniae isolates isolated in China between 2011 and 2012 were found to be 0.5 and 1 mg/l, respectively. In the study of Yin et al.^[7] conducted in 2017, CZA MIC₉₀ values in E. cloacae and K. pneumoniae were reported as ≥32 and 4 mg/l, respectively, and it was suggested that the decrease in CZA susceptibility rates in these species might be associated with the rapid increase in the spread of especially NDM-1 producing isolates in carbapenem-resistant Enterobacteriaceae.

One of the limitations of our study was the low number of isolates tested. In addition, the clinical sample diversity in the isolates was limited. In addition, another limitation of our study was that only MDR K. pneumoniae isolates were tested.

Conclusion

All of the MDR K. pneumoniae isolates examined in our study were found to be resistant to other beta lactam and beta lactamase inhibitors as well as to carbapenems. In vitro antibacterial activity of CZA against K. pneumoniae was found to be higher than C/T. While CZA was found to be very effective in OXA-48 and KPC producing MDR K. pneumoniae isolates, it was determined that the efficiency of C/T was low. The ability of avibactam to inhibit KPC type carbapenemases suggests that the CZA combination will be an effective option in isolates producing KPC type carbapenemase. However, CZA was found to be ineffective in isolates producing NDM type carbapenemase in our study. More comprehensive studies are needed to determine the role of these two combined treatment options in the treatment of infections caused by MDR Gram-negative bacteria.

Ethics

Ethics Committee Approval: Approval was obtained from Sakarya University Faculty of Medicine Ethics Committee for this study (ethics committee approval no: 71522473/050.0104/75, date: 16.08.2017).

Informed Consent: Informed consent form was not received due to the retrospective nature of the study.

Peer-review: Externally and internally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices: H.A.T., M.K., Concept: H.A.T., Ö.A., Design: H.A.T., Ö.A., Data Collection or Processing: H.A.T., M.K., Ö.A., Analysis or Interpretation: H.A.T., M.K., Literature Search: H.A.T., M.K., T.D., M.A., Ö.A., Writing: H.A.T., M.K., T.D., M.A., Ö.A.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

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Evaluation of In Vitro Activity of Ceftolozane-tazobactam and Ceftazidime-avibactam Against Carbapenemase-producing Multi-drug Resistant Klebsiella pneumoniae Isolates