Characterization of Tigecycline Resistance Among Tigecycline Non-susceptible Klebsiella pneumoniae Isolates From Humans, Food-Producing Animals, and in vitro Selection Assay.

Mohaddeseh Moghimi,Mehri Haeili,Hanieh Mohajjel Shoja

doi:10.3389/fmicb.2021.702006

Abstract

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR, acrR, rpsJ, tetA, and mgrB (for colistin resistance) genes were sequenced. The presence of tetX, tetX1, and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4–32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (blaOXA–48, blaNDM–1, and blaNDM–5). tetX/X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR, and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates.

Highlights

Antibiotic resistance is rising to dangerously high levels in both human and veterinary medicine
Among the 1,430 samples taken from healthy broilers, six TGC-resistant (TGC-R) bacteria were detected, all corresponding to K. pneumoniae
One TGC-R isolate (AK513, TGC minimum inhibitory concentrations (MICs) = 8 mg/l) was included from our previous work (Pishnian et al, 2019), which was obtained from a turkey during the screening for colistin-resistant bacteria (Table 2)

Summary

Introduction

Antibiotic resistance is rising to dangerously high levels in both human and veterinary medicine. The use of human antibiotics in food animals as growth promoters or prophylactic agents has been identified as a significant contributing factor to increasing antimicrobial resistance (Van et al, 2020). Klebsiella pneumoniae is among the most problematic pathogens that have acquired much public health concern due to developing resistance to most clinically important antibiotics. TGC is the first member of the novel class of glycylcyclines with expanded-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria. It is approved by the Food and Drug Administration (FDA) for use in complicated skin and skin structure infections, complicated intra-abdominal infections, and community-acquired bacterial pneumonia (Beabout et al, 2015). TGC acts by inhibition of bacterial protein synthesis and has the ability to evade the classical mechanisms mediating resistance to tetracyclines including ribosomal protection and active efflux mediated by Tet proteins (Livermore, 2005)

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