*Article* **Determination of Mutational Timing of Colistin-Resistance Genes through** *Klebsiella pneumoniae* **Evolution**

**Jenna M. Kuhn and Yuanpu Peter Di \***

Department of Environmental and Occupational Health, School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA

**\*** Correspondence: peterdi@pitt.edu; Tel.: +1-(412)-624-8718

**Abstract:** The emergence and dissemination of carbapenem-resistant *Klebsiella pneumoniae* (KP), one of the carbapenem-resistant *Enterobacteriaceae* (CRE), is now an emerging cause of antibiotic-resistant nosocomial infections associated with high rates of morbidity and mortality. Colistin, or polymyxin E, is a last-resort peptide antibiotic used to treat multidrug-resistant (MDR) Gram-negative bacterial infections including KP. Unfortunately, resistance to colistin is rising with increasing use in the clinical setting. Although clinical evidence links certain mutations to colistin resistance (COL-R) in KP, the origination and association of the mutations remain unclear. We hypothesize that the timing of COL-R mutations influences the development and progression of KP resistance to colistin. We performed planktonic and biofilm in vitro experimental evolutions of KP strain ATCC 43816 under increasing colistin concentrations to characterize the temporal regulation of critical COL-R mutations throughout COL-R progression. The resistance generation and mutation profiles of independently evolved bacterial populations with different lifestyles were compared. Genes with various functions theorize the timeline in which key mutations are generated and their roles in the progression of COL-R. Our results aim to advance the research and development of effective therapeutics to treat MDR bacterial infection as the dissemination of CRE continues to be a severe public health threat.

**Keywords:** *Klebsiella pneumoniae*; colistin; antimicrobial resistance; mutation timing; evolution

**1. Introduction**

The emergence of carbapenem-resistant Enterobacterales (CRE) has become a global public health threat with high mortality rates of infection and limited available antimicrobial treatment options [1]. According to the US Centers for Disease Control and Prevention 2019 Antibiotic Resistance Threats Report, CRE infections led to approximately 13,100 hospital incidents and 1100 infection-related deaths, with average case numbers remaining steady between 2012 and 2017 [2]. Additionally, the worldwide dissemination of extended-spectrum β-lactamases (ESBLs) in species of *Enterobacteriaceae* and *P. aeruginosa* has facilitated resistance to a number of β-lactam antibiotics, including carbapenems, cephalosporins, monobactams, and penicillins, as well as recent detection of resistance to aztreonam and oxyimino-cephalosporins [3,4]. A pathogen of urgent concern of the *Enterobacteriaceae* family, *Klebsiella pneumoniae* (KP), is a one of six nosocomial ESKAPE pathogens (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter* species), noted for its virulence and high potential for multidrug resistance (MDR) [5]. KP is characterized as an encapsulated, Gram-negative, nonmotile, facultative anaerobic pathogen acquired in community or healthcare settings that may cause pneumonia, urinary tract infection, soft-tissue infection, bacteremia, and meningitis, especially in immunocompromised individuals [6]. The recent emergence and rising prevalence of carbapenem-resistant hypervirulent KP (CR-hvKP) has several concerning clinical impacts due to high resistance and pathogenicity, high mortality, production of multiple carbapenemases, and gut colonization, facilitating further resistance

**Citation:** Kuhn, J.M.; Di, Y.P. Determination of Mutational Timing of Colistin-Resistance Genes through *Klebsiella pneumoniae* Evolution. *Pharmaceutics* **2023**, *15*, 270. https://doi.org/10.3390/ pharmaceutics15010270

Academic Editor: Ivana Cacciatore

Received: 16 November 2022 Revised: 16 December 2022 Accepted: 4 January 2023 Published: 12 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

dissemination [7]. Due to rising numbers of identified ESBLs and the global spread of CR-hvKP, there are limited treatment options available.

Colistin, or polymyxin E, is a last-resort antibiotic reserved for treating complex CRhvKP infections. Polymyxins are cationic cyclic polypeptides that belong to a family of antimicrobial peptides (AMPs), which are bactericidal components of the host innate immune system present in plant and animal species, as well as some bacteria and fungi. AMPs are usually amphipathic, cationic, and approximately 15–30 amino acids in length; they impose their activity at the cell membrane of bacteria [8]. The two dominant polymyxins used clinically to treat Gram-negative bacterial infections are polymyxin B and polymyxin E (Figure 1) [9,10]. AMPs challenge the development of drug resistance due to their diverse killing mechanisms and low specificity for a given target in host cells [11].

**Figure 1.** Chemical structure of polymyxins B and E. The primary polymyxins used clinically to treat Gram-negative infections are the cyclic polypeptides Polymyxin B and E. Both polymyxins enact their activity through binding to negatively-charged lipopolysaccharide and disruption of outer membrane permeability. The two-dimensional chemical structures and formulas for polymyxin B and polymyxin E displayed in this figure were obtained from PubChem, with CIDs 4868 and 5311054, respectively.

Colistin is a cationic polypeptide that exerts its activity by binding to anionic regions of lipopolysaccharide (LPS), leading to permeabilization of the cell envelope, cell leakage, and cell death [12]. Since its displacement by other antimicrobials four decades prior, colistin has shown significant activity against KP, *P. aeruginosa*, *A. baumannii*, and other Gram-negative pathogens with low initial resistance levels [13]. However, with increased use in the clinical space, rates of colistin resistance have been on the rise through several main mechanisms, including chromosomal mutations in genes responsible for disrupting the cationic charge of LPS (PhoP/PhoQ and PmrA/PmrB two-component regulatory systems) and MgrB, a transmembrane regulator of PhoP/PhoQ signaling, as well as plasmid-mediated colistin resistance (*mcr-1-10*) [14,15]. It has also been shown that increased production of capsular

polysaccharide hinders interactions between colistin with the cell membrane of bacteria, facilitating resistance [14]. While the primary mechanisms of COL-R have been identified, the relationship among resistance generation timing, population frequency, and interactions of mutations facilitating the onset and progression of COL-R has not been thoroughly investigated. The demand for further development of effective antimicrobials against the growing public health threat of MDR infections continues to grow. Now, even last-resort treatments are showing high rates of resistance.

In particular, biofilm-associated infections comprise ~65% of all bacterial infections in the clinical setting and present a serious challenge to the healthcare community due to their diversity and innate defense mechanism to protect against antimicrobials [16,17]. There are several mechanisms via which biofilms may resist antimicrobial agents. The exopolysaccharide matrix of biofilm environments may hinder penetration of negatively charged antimicrobials such as aminoglycosides [17]. Furthermore, microcolonization within the biofilm leads to waste accumulation and fluctuation of pH and CO2 and O2 partial pressures, consequently disrupting antimicrobial activity [17]. Additionally, regulation of efflux pumps, expression of antimicrobial chelating enzymes, and quorum sensing are other biofilm-associated resistance mechanisms [17]. KP biofilms may lead to invasive, chronic infections in the urinary, gastrointestinal, or respiratory tracts through cell adhesion to a surface, colony formation, biofilm maturation, and cell detachment [18]. Here, we consider both planktonic and biofilm KP lifestyles in our experimental evolution methods to better understand the timing of critical resistance mutations and their likely impact on the progression of COL-R.
