Evaluation of in vitro activity of ceftazidime/avibactam among carbapenamase producing Klebsiella pneumoniae from clinical isolates in tertiary care hospital

The emergence of multidrug resistant organisms pose a great challenge to global health. Carbapenem group of drugs have been used as treatment therapy for these multidrug resistant organisms. Ceftazidime-avibactam is a newer combination consisting of ceftazidime, a 3rd generation anti pseudomonal cephalosporin and avibactam, a beta lactamase inhibitor. Avibactam helps in restoring the antibacterial activity of ceftazidime against carbapenamase producing Enterobacterales. Ceftazidime-avibactam (CAZ-AVL) provides us a valuable strategy to combat carbapenem resistant infections especially in Enterobacterales. The objective of the study was to find out carbapenem resistant strains of Klebsiella pneumoniae from clinical isolates and to detect the carbapenamase activity by phenotypic and genotypic detection methods. Carbapenamase detection from the clinical isolates was done by meropenem hydrolysis test (mCIM). The resistant strains were evaluated for the activity of ceftazidime/avibactam by Estrip method. Molecular analysis of all the carbapenem resistant strains were done for blaOXA48 and blaKPC. Among 2500 isolates which were subjected to study from January-June 2022, 306 isolates grew Klebsiella pneumoniae among which 34 (11%) isolates were found to be multidrug resistant organisms (MDRO). Modified carbapenem inactivation method (mCIM) was used for carbapenamase activity. Ceftazidime/avibactam susceptibility was done using Estrip. 28(82.4%) isolates were found susceptible (≤8/4ug/ml) and 6(17.6%) isolates were found resistant (≥16/4ug/ml). Among CAZ/AVL resistant strains blaKPC was more prevalent than blaOXA48. In our study it was found that prevalence of OXA-48 was more than KPC among the resistant strains of Klebsiella pneumoniae and the susceptibility of ceftazidime/avibactam was more in Enterobacterales.

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Introduction

Resistance in Gram-negative organisms have been emerging these years. The resistance is probably due to indiscriminate use of antibiotics and prolonged hospital stay. Multidrugresistant Gram-negative organisms are treated with carbapenems especially in ICU (intensive care units) in most of the hospitals. Because of the rampant use, we are now facing problems with carbapenem resistance [1] . The resistance to carbapenem group of antibiotics are due to over expression of ESBL (extended spectrum of beta-lactamase), associated with poring loss, production of carbpenemhydrolysing enzymes like KPC (class A) Amp C beta-lactamase, MBL (class B metallo-beta-lactamase) and Carbapenamase like OXA-48 Class D) [2] . Among these carbapenamase enzymes prevalence of blaOXA-48 has been widely observed in Belgium, France and Spain [3] . The primary report of Klebsiella pneumoniae carbapenemase (KPC)-in K. pneumoniae was reported North Carolina in 2001 [4] . In 2005 it was noted that this KPC producing carbapenamase was found to be spreading worldwide [5] . In addition, OXA-48-like and New Delhi metallo-beta-lactamases are the other carbapenemases of clinical significance in Enterobacterales. In India NDM-1 (New Delhi beta-lactamase) is the most frequently identified carbapenamase [3] . Besides KPC, it was observed that prevalence of OXA-48 was higher in parts of Southeast Asia and India [6] .

Study showed that ceftazidime/avibactam showed wide range of in vitro activity against KPC-producing Enterobacterales even in the presence of other hydrolyzing enzymes. Recent data from US, showed effectiveness of ceftazidime/avibactam against 120 KPC producers, even in the presence of other β-lactamases which has the ability to hydrolyze ceftazidime [7] . The activity of OXA-48 by ceftazidime/avibactam is complex due to its inability to hydrolyze ceftazidime. However, few studies have proved the coproduction of OXA-48-like enzymes with extendedspectrum β-lactamases (ESBLs) [8] . Hence, avibactam is necessary to restore the activity of ceftazidime when there is coproduction of OXA-48 and ESBL [5] . The mortality rates lies between 20% and 50% in critically ill patients with invasive infections due to carbapenem-resistant Enterobacterales (CRE) [9,10] . Avibactam is a 𝛽-lactamase inhibitor which is active against class C enzymes, many of class An enzymes (including KPCs), and few class D (OXA) carbapenemases [11] . The object of this study was done to find out the in vitro activity of ceftazidime/avibactam among MDR Klebsiella pneumoniae from various clinical samples in a tertiary care centre.

Materials and Methods

The prospective study was done at Saveetha Institute of Medical and Technical Sciences, Chennai was done during the period from January to June 2022. Ethical clearance was obtained from the institution. Statistical analysis was done using SPSS software. Descriptive and inferential statistical analysis was done for the analysis. All the samples from inpatients of various departments were used for the study. All samples from outpatients were excluded in our study. The samples included urine, blood, exudate, body fluids CSF (cerebrospinal fluid), tissue samples, etc. All the samples were processed after preliminary identification and were streaked on MAP (MacConkey agar), CAP (Chocolate agar) and BAP (blood agar), CLED (Cysteine lactose electrolyte deficient) agar depending on the samples and were processed in VITEK 2 system for organism identification and antibiotic susceptibility testing. Samples which were resistant to carbapenem group of drugs were taken for further study. Carbapenemase detection was done by mCIM as given by CLSI 2022 [12] . mCIM Method: 1ul loopful of bacteria from an overnight blood culture was emulsified into 2ml of TSB (Trypticase Soy Broth). The sample was vortexed for 10-15 seconds. 10 ug meropenem disc was dispensed in the tube and the disc was completely immersed in broth and incubated at 37 deg C for 4 hours. On a MHA (Muller Hinton agar plate) ATCC (American type culture collection) strain E. coli 25922 was streaked by lawn culture. The meropenem disc was removed making sure that the excess fluid was removed and kept at the centre of MHA. The plate was incubated overnight and looked for a zone of inhibition. If the meropenem disc was hydrolyzed there would have been no zone of inhibition. Carbapenamase activity was detected by this method. It is much more sensitive than the modified Hodge test. If zone of inhibition was ≥ 19 mm it was considered negative.

E strip by Ceftazidime and Ceftazidime + avibactam

E strip was done using ceftazidime + avibactam to assess the in vitro activity of these drugs on MHA plate. Susceptibility was observed when there was zone of inhibition of ≤ 8/4 ug/ml and for resistance organisms it was ≥16/4 ug/ml. PCR detection by gel electrophoresis. PCR detection of the resistant strain was done by gel electrophoresis. The genes detected were bla OXA48 and bla KPC.

The primer pair used to detect the OXA-48 gene was OXA-48F (5ʹ-GCTTGATCGCCCTCGATT-3ʹ) and OXA-48R (3ʹ-GATTTGCTCCGTGGCCGAAA-5ʹ)

The primer for KPC blaKPC (forward primer 5′-ATGTCACTGTATCGCCGTCT-3′; reverse primer 5′-CCTAAATGTGACAGTGGTTGG). The extraction procedure for both the genes were as follows Plasmid DNA isolation: The plasmid DNA from Klebsiella pneumoniae was isolated using the QIAprep Spin MiniPrep kit. The pellet was obtained by centrifuging about 5 ml of overnight-grown bacterial culture at 8000 rpm for 3 minutes at room temperature. The pellet was suspended in 250 microlitres of Buffer P1 and transferred to a micro-centrifuge tube. About 250 microlitres of buffer P2 was added to the suspension and mixed thoroughly by inverting the tubes 4-6 times until the solution becomes clear. To the lysate about 350 microlitres of buffer N3 was added and immediately mixed by inverting the tubes 4-6 times. The lysate was spun at 13000 rpm for 10 mins at room temperature. About 800 microlitres of cleared lysate was added to the QIAPrep 2.0 spin column by pipetting. The tubes were then spun for 30-60 seconds. The flow-through was discarded. The column was washed by adding 0.5 mL of Buffer PB. The tubes were spun for 30-60 seconds and the flow through was discarded. The column was spun for an additional time of 1 minute to remove any residual wash buffer. The QIAPrep 2.0 column was placed in a sterile 1.5 mL tube. About 50 microliters of Buffer EB was added to the centre of the column. The tubes were incubated at RT for 1 minute and spun for 1 min at 12000 rpm. About 5 µl of eluted plasmid DNA was loaded onto 0.7% agarose gel. The plasmid DNA was visualized using a UV trans-illuminator.

Results

During the period of 6 months (January 2022-June 2022), we received 2500 samples from various departments. The samples included sputum, CSF, urine, blood, and sterile fluids. Among those 2500 isolates, 306 isolates grew Klebsiella pneumoniae. Out of the 306 isolates, 34 (11%) isolates were found to be multidrug-resistant organisms (MDRO). These MDR Klebsiella pneumoniae were isolated from patients of different age groups (11.8% were <40 years, 23.5% were between 40-50 years, and 64.7% were >50 years of age). MDR Klebsiella pneumoniae were isolated more from men (70.6%) compared to women (29.4%). In our study, the clinical wards (61.8%) had more MDR Klebsiella pneumoniae compared to that of various Intensive Care Units {Medical ICU (32.8%), Surgical ICU (2.9%) and Respiratory ICU (2.9%), (Table-1). Out of 34 MDR Klebsiella pneumoniae strains, they were isolated more from exudate samples (41.2%) like wound swab, pus, tissue followed by urine samples (32.4%), respiratory samples (14.7%) and least in blood samples (11.8%).

Phenotypic and genotypic tests were performed amid the MDR Klebsiella pneumoniae isolated from various clinical samples to look for the carbapenemase activity. Modified carbapenem inactivation method (mCIM) was carried out and all the 34 (100%) isolates were mCIM positive. (Fig 1) Ceftazidime/avibactam susceptibility for the MDR strains were tested using Estrip, (Figure 2) 28(82.4%) isolates were found susceptible (≤8/4 ug/ml) and 6(17.6%) isolates were found resistant (≥16/4 ug/ml). The results of the molecular analysis of the resistant strains of MDR Klebsiella pneumoniae showed blaOXA-48 positive 22(64.7%) and negative 12(35.3%) and blaKPC positive 14(41.2%) and negative 20(58.8%).

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Ceftazidime-avibactam susceptibility

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Positive mCIM method

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Discussion

This study was focused to find out the in vitro activity of ceftazidime/avibactam in MDR Klebsiella pneumoniae. We encounter MDR organisms of Klebsiella pneumoniae in clinical wards compared to that of ICUs. Our study was focused to find out the in vitro activity of ceftazidime/ avibactam among MDR strains of Klebsiella pneumoniae. In our study, there were 34 multidrug-resistant (11%) strains out of 306 strains of Klebsiella pneumoniae. These MDR strains which were beta carbapenem-resistant were responsible for more mortality [13] . Emphasizing that in 2017 WHO (World Health Organization) declared carbapenemresistant Enterobacterales (CRE) as first critical pathogens. Ceftazidime-avibactam (CZA) is a newer combination of a third-generation cephalosporin with second-generation betalactamase inhibitor [14] . The antibacterial activity against carbapenamase-producing Enterobacterales, is restored through the combination of ceftazidime-avibactam [11] . In our study out of 34 MDR strains 28 strains showed susceptibility to ceftazidime/avibactam. Only 6 strains were resistant to ceftazidime/avibactam. In Young et al. [14] study, he observed that 21 strains out of 386 isolates in Klebsiella pneumoniae were resistant to Ceftazidime/avibactam. It was observed that mortality rate was lower with patients on treatment with ceftazidime and avibactam when used in combination with ceftazidime. This was observed in Tumbarello et al. study where the mortality observed was 37% [15] . The mutations in the omega loop of the KPC enzyme leads to enhanced ceftazidime hydrolysis. This cannot be inhibited by avibactam resulting in resistance to ceftazidime/avibactam. The resistance could be due to mutation in tyrosine for aspartic acid substitution at omega

International Journal of Biology Sciences https://www.biologyjournal.net loop of the KPC enzyme at position 179 [16] . The resistance in Klebsiella pneumoniae to ceftazidime/avibactam is due to mutation in bla3KPC Shields RK et al. study [17] . Besides carbapenemase activity, decreased outer membrane permeability and increased efflux pump mechanism can also lead to CZA resistance in Pseudomonas aeruginosa as discovered by Winkler and colleagues. Few other studies also described the overexpression of AmpC as a resistance mechanism for CZA, especially in Pseudomonas aeruginosa [18][19][20] . Molecular analysis was done for all 34 strains of MDR Klebsiella pneumoniae. We looked for presence of blaKPC and blaOXA-48 genes. In HA Hakeam, H Alsahli, L Albabtain et al. blaOXA-48 was observed in 31 isolates out of 50 isolates (66%). Our molecular analysis showed the presence of blaOXA-48 more than blaKPC. 22 (64.7%) strains were OXA-48 positive 14(41.2%) were KPC positive and 11 strains had both OXA and KPC positive. It is similar to Oteo et al. [21] study in Spanish hospitals which showed 90.5% of Klebsiella pneumonia harboring OXA-48. Azimi et al. study [22] , reported first case of OXA 48 in Iran. In Haekaem et al. study [23] blaOXA 48 was observed in CRE Enterobacterales which is similar to our study. We analysed the presence of OXA and KPC in ceftazidime/avibactamresistant strains. In this study, among the 6 ceftazidime/ avibactam-resistant strains, 2(33.3%) strains harbored both OXA-48 and KPC, Only 3(50%) strains were KPC positive which OXA-48 negative were. In our study, among ceftazidime/avibactam resistant strains KPC was more prevalent than OXA 48.1(16.6%) strain had both OXA-48 and KPC negative. Here the mechanism could have been due to impermeability or porin loss. In Azimi et al. study [22] also, out of 28 MDR strains of Klebsiella pneumoniae 27 strains showed presence of blaOXA48 and one strain was negative which is similar to our study. Interestingly, De la Calle et al. study observed that there was the absence of resistance to ceftazidime-avibactam during the treatment of infections due to CRE, inhabiting the blaOXA-48 gene [24] . It has been observed that ceftazidime/avibactam is a promising drug, especially in ICUs.

Conclusion

Though the sample size was small, it clearly indicates ceftazidime/avibactam is an ideal drug for Enterobacterales especially in intensive care units. Ceftazidime-avibactam, a beta-lactamase antibacterial agent is definitely an important drug for the management of MDR Gram-negative pathogens. Especially in the Indian scenario, because of the cost factor limited data is currently available to support its clinical efficacy. Implementation of antimicrobial stewardship and strict infection control practices can help preserve the susceptibility of the ceftazidime-avibactam combination.

Acknowledgement

AcknowledgementWe would like to acknowledge, 1. Statistician-Niharika S, Ashika R, Swathi Gupta for creation of Tables, Listing, and Graphs (TLG) and Statistical Analysis Reporting (SAR) and Dr. K.P. Suresh, Ph.D. (Biostatistics) for reviewing methodology and results. 2. Dr. Vijayashree Priyadarshini, Associate Professor (clinical genetics), centre for Cellular and Molecular Research, Saveetha Dental College and Hospital, Chennai. For molecular analysis 3. Pfizer Pharmaceuticals, India for providing E strips of Ceftazidime/avibactam. 4. Professors and technicians of Saveetha Institute of Medical and Technical Sciences, Chennai.

Figures and tables from the paper

Ward/ICU	No. of Patients	%
WARD	21	61.8
MICU	11	32.4
SICU	1	2.9
RICU	1	2.9
Total	34	100.0

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Frequency of distribution of patients studied

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Distribution of OXA-48 and KPC genes among MDR Klebsiella pneumoniae

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