Minimum inhibitory concentration of Ceftazidime-Avibactam against Clinical isolates of XDR Salmonella enterica Serovar Typhi


 Background: Typhoid fever is a major healthcare problem in low and middle-income countries. The emergence of extensively drug-resistant (XDR) typhoid has further narrowed down the way to already limited therapeutic options. WHO has listed S.Typhi amongst the priority pathogens against which new treatment options should be discovered.Objective: To determine In-vitro activity of Ceftazidime-avibactam against clinical isolates of XDR S.Typhi.Study design: This is a cross-sectional studyPlace and duration of the study: The Department of Microbiology, University of health sciences, Lahore from January to June 2021.Methodology: Antimicrobial susceptibility was performed initially by Kirby Bauer disc diffusion method for 150 of XDR Salmonella enterica Serovar Typhi and MICs of all the recommended antibiotics was determined by VITEK 2 (bioMérieux) fully automated system using Clinical Laboratory Standard Institute (CLSI) 2021 guidelines. MICs by the E-test method were determined for Ceftazidime-avibactam and Azithromycin only. All tests were done in duplicate.Results: All 150 (100%) isolates were sensitive to Ceftazidime-avibactam by disc diffusion and E-test methods. Out of 150 isolates 8(5.3%) were having high MICs against Azithromycin.Conclusions: Ceftazidime-avibactam can be used wisely to treat ESBL producing XDR typhoid fever cases especially in countries like Pakistan where Typhoid fever is endemic and majority of isolates are extensively drug resistant.


Introduction
Typhoid fever remains a signi cant public health threat in low and middle-income countries. According to the most recent estimates, between 11 and 21 million cases and 128 000 to 161 000 typhoid-related deaths occur annually worldwide [1]. Antimicrobial resistance in Salmonella can be associated with horizontal transference of antibiotic resistant genes characteristically found on mobile genetic elements among Salmonella strains and other Enterobacteria or by clonal spread of antimicrobial drug resistant serovars that are particularly nominated in worldwide dissemination [2]. Since November 2016, a large proportion of ceftriaxone-resistant cases have been identi ed in the province of Sindh. Similar case was also identi ed in the United Kingdom from a traveler returning from Pakistan. These S.Typhi strains are resistant to chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, uoroquinolones, and thirdgeneration cephalosporins. This new strain has been designated as XDR (Extensively drug resistant) leaving behind azithromycin and carbapenems, the only treatment options [2].This Pakistan outbreak caused by an XDR H58 strain of S.Typhi should be regarded as a clarion call that noti es public health authorities globally that we are rapidly approaching a scenario where the acquisition of one more resistance might result in an S. Typhi pathogen that is, in practical terms, virtually untreatable in most developing countries. Now is the time for global action to prevent a gathering storm from becoming a perfect storm and an enormous public health crisis. Ceftazidime is a third generation cephalosporin antibiotic useful for the treatment of a number of bacterial infections. Speci cally it is used for joint infections, meningitis, pneumonia, sepsis, urinary tract infections, malignant otitis externa, Pseudomonas aeruginosa infection, and vibrio infection [3]. Like all other third generation cephalosporins, it is active against Salmonella enterica serovars including S.Typhi. But recent emergence of XDR S.Typhi has knocked this drug out of the arsenal because of ESBL production. Avibactam is a non-β-lactam β-lactamase inhibitor developed by Actavis.
A new drug application for avibactam in combination with ceftazidime (branded as Avycaz) was approved by the FDA on February 25, 2015, for treating complicated urinary tract (cUTI) and complicated intra-abdominal infections (cIAI) caused by antibiotic resistant-pathogens, including those caused by multi-drug resistant Gram-negative bacterial pathogens [4].Recently approval has been given for bacterial pneumonia and ventilator associated pneumonia caused by multi drug resistant pathogens.
Ceftazidime/avibactam has been shown to be highly active against Enterobacteriaceae in in-vitro studies, inhibiting a broad spectrum of β-lactamases. In a study, the activities of ceftazidime-avibactam and comparator antimicrobial agents were tested against 20,709 clinical Enterobacteriaceae isolates collected in U.S. hospitals during the period from 2011 to 2013. Overall, 99.9% (20,698 of 20,709) of Enterobacteriaceae strains were inhibited at a ceftazidime-avibactam MIC of 8 µg/ml or less, which is the ceftazidime-avibactam-susceptible breakpoint. This study included 284 CTX M−15 producing isolates and 107 CTX M−14 producers. The potent Gram-negative spectrum of activity of ceftazidime-avibactam, including activity against resistant organisms, demonstrates that it warrants further study in di cult to treat serious infections where resistant gram negative bacteria may occur [6]. Ceftazidime-avibactam activity has been reported in Salmonella enterica having MIC 50/90 equal to 0.25/0.5 with range of MIC ≤ 0.03-0.5 and 100% isolates were sensitive to this combination [7].

Study Design
This was a Cross-sectional study.

Setting
This study was conducted at The Department of Microbiology, University of Health Sciences, Lahore.

Sample Collection:
A total of 150 positive blood cultures meeting the inclusion criteria were collected from blood different tertiary care hospitals of Lahore.

Bacterial identi cation
The isolates were cultured and puri ed on Tryptic Soya agar. The blood culture bottle detected as positive was sub-cultured onto Blood agar (Oxoid, UK) and MacConkey agar (Oxoid, UK) incubated at 35-37 °C. The isolate identi cation was initially performed by Gram-staining. Biochemical identi cation was done by using Analytical Pro le Index-20 Enterobacteriacae system (BioMerieux, France). France) and VITEK2 (bioMérieux) consistent with the manufacturer instructions. S.Typhi was con rmed by agglutination with genus-and serotype-speci c antisera ( This study was conducted using the bacterial strains which were isolated for diagnostic and treatment purpose. Moreover, the study was completely anonymous, any demographic data or identi able information was not obtained therefore informed consent was not required for such type of study according to the local legislation.

Results
In this study, a total of 150 XDR S. Typhi isolates were included which were collected over a period of 6 months from different tertiary care hospitals of Lahore.

Discussion
Typhoid fever is a signi cant health problem for countries like Pakistan. Emergence and spread of XDR S.Typhi across Pakistan has generated a huge problem for the control and prevention of Typhoid fever. These S.Typhi strains are resistant to chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, uoroquinolones, and third-generation cephalosporins. With very limited treatment options already available emergence of Azithromycin resistance would create further problem. There is dire need to look for new therapeutic options as urged by WHO (World health organization).
Ceftazidime-avibactam has shown promising results against MDR and XDR pathogens producing ESBLs in previous studies. In a recent study, a total of 7051 Enterobacterale isolates and 2032 Pseudomonas aeruginosa isolates from hospitalized patients in Australia, Japan, South Korea, Malaysia, the Philippines, Taiwan and Thailand were studied. More than 90% of all Enterobacterales isolates, including the ESBL-positive, carbapenemase-negative and the carbapenemase-positive, MBL-negative were susceptible to amikacin and ceftazidime-avibactam [9].Ceftazidime-avibactam and comparators were tested by reference broth microdilution against 372 Gram-negative bacilli collected from 11 teaching hospitals in China in 2011 and 2012. Avibactam potentiated the activity of ceftazidime against organisms with combinations of ESBLs, AmpCs, and KPC-2 [10].
In another study Ceftazidime-avibactam was tested against 57 well-characterized Gram-negative strains producing beta lactamases from all molecular classes. Avibactam lowered ceftazidime MICs up to 2,048fold against AmpC extended-spectrum beta lactamase (ESBL), and KPC-2 producing Enterobacteriaceae or Pseudomonas aeruginosa [11].
In another study conducted in Spain, Ceftazidime-avibactam was very active against ESBL producing Enterobactarales. Escherichia coli had MIC 90 of 0.25 mg/liter and ESBL producing Klebsiella pneumonia had MIC 90 of 0.5 mg/liter, Ceftazidime-resistant AmpC-producing species had MIC 90 of 1 mg/liter [12].
Furthermore, in a large US focused surveillance program conducted in 2012, ceftazidime-avibactam had antimicrobial activity against 99.8% of Enterobacteriaceae including those with an ESBL phenotype [12].
Ceftazidime-avibactam can be used as a treatment option in ESBL producing XDR S.Typhi isolates sparing Carbapenems and Azithromycin. This will reserve the use of these drugs in non-responding or di cult to treat cases without signi cantly creating resistance by overuse. Azithromycin resistance has already been reported from several parts of the world especially typhoid endemic countries like India,Nepal,and Bangladesh [13].Several cases have been reported from Pakistan too [14].

Conclusion
Azithromycin resistance in XDR S.Typhi is on the rise and it may pose a signi cant threat to the clinicians especially where XDR S.Typhi is endemic now. Antimicrobial stewardship especially in the post-COVID-19 era is crucial to prevent the development of resistance to this limited oral choice against XDR typhoid. There is vital need to look for new treatment options. Ceftazidime-avibactam has shown promising results in this in-vitro study and it can be a likely candidate for clinical trials.

Declarations Ethical approval
The study was ethically approved by the "Ethical Review Board" under reference number UHS/REG-19/ERC/398 from University of health sciences, Lahore, Pakistan.

Con ict of interest
The authors declare no competing interests negative bacilli collected in 2011 and 2012 from 11 teaching hospitals in China. Antimicrob. Agents Chemother, 58 (3) Table: 1