Next Article in Journal
Molecular Genetic Understanding of Photoperiodic Regulation of Flowering Time in Arabidopsis and Soybean
Next Article in Special Issue
Whole Genome Analysis of 335 New Bacterial Species from Human Microbiota Reveals a Huge Reservoir of Transferable Antibiotic Resistance Determinants
Previous Article in Journal
Generation of Lasso Peptide-Based ClpP Binders
Previous Article in Special Issue
Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 1
10.3390/ijms23010449
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Antibiotic Heteroresistance in Klebsiella pneumoniae

by
Karolina Stojowska-Swędrzyńska
,
Adrianna Łupkowska
,
Dorota Kuczyńska-Wiśnik
and
Ewa Laskowska
*
Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(1), 449; https://doi.org/10.3390/ijms23010449
Submission received: 13 December 2021 / Revised: 28 December 2021 / Accepted: 29 December 2021 / Published: 31 December 2021
(This article belongs to the Special Issue Molecular Mechanism and Detection of Anti-microbial Resistance in Bacteria)
Browse Figures
Versions Notes

Abstract

:
Klebsiella pneumoniae is one of the most common pathogens responsible for infections, including pneumonia, urinary tract infections, and bacteremias. The increasing prevalence of multidrug-resistant K. pneumoniae was recognized in 2017 by the World Health Organization as a critical public health threat. Heteroresistance, defined as the presence of a subpopulation of cells with a higher MIC than the dominant population, is a frequent phenotype in many pathogens. Numerous reports on heteroresistant K. pneumoniae isolates have been published in the last few years. Heteroresistance is difficult to detect and study due to its phenotypic and genetic instability. Recent findings provide strong evidence that heteroresistance may be associated with an increased risk of recurrent infections and antibiotic treatment failure. This review focuses on antibiotic heteroresistance mechanisms in K. pneumoniae and potential therapeutic strategies against antibiotic heteroresistant isolates.

1. Introduction

Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile bacterium commonly present in the environment and primarily associated with community-acquired pneumonia in patients with alcohol use disorder or diabetes mellitus [1,2]. In healthy humans, K. pneumoniae colonizes mucosal surfaces of the oropharynx, nasopharynx, and gastrointestinal tract [1,2]. Classical K. pneumonia (cKP), naturally resistant to ampicillin, carbenicillin, and ticarcillin due to the production of a chromosomal penicillinase (SHV-1), is becoming increasingly multidrug-resistant via the acquisition of resistance determinants carried on the chromosome or mobile elements (plasmids and transposons) [2,3,4]. Multidrug-resistant (MDR) cKP is one of the main factors responsible for nosocomial infections, including pneumonia, urinary tract, and bloodstream infections. MDR cKP producing extended-spectrum β-lactamases (ESBL) and carbapenemases are responsible for high mortality rates (40–50%), mainly among critically ill and immunocompromised patients [3]. The continuous accumulation of antibiotic-resistant genes in cKP has resulted in the emergence of extensively drug-resistant (XDR) and pandrug-resistant (PDR) strains with “super resistome”, which protects cKP against all available antimicrobials [5,6,7]. Over the last few years, an increasing number of hypervirulent K. pneumoniae (hvKP) isolates expressing a hypermucous phenotype have been repeatedly reported. The hvKP isolates possess a thick capsule and are more resistant to phagocytosis, complement-, and neutrophil-mediated activities than cKP [4]. It has been suggested that hvKP strains could survive within macrophages and neutrophils. Moreover, hvKp can use neutrophils as vehicles, in line with the “Trojan horse” infection mechanism, leading to disseminated infections [4]. Moreover, hvKP causes highly invasive infections in healthy and immunocompromised individuals, such as pyogenic liver abscess, endophthalmitis, and meningitis, [2,3,4,8,9,10,11,12]. The convergence of resistance and hypervirulence factors on one or multiple coexisting plasmids has resulted in the evolution of MDR and XDR hvKP isolates, which are now recognized as a severe threat to public health [4,13].
Due to life-threatening or deadly infections, K. pneumoniae was classified as the ESKAPE pathogen, an acronym for the Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. The ECAPE pathogens cause most nosocomial infections and are capable of “escaping” conventional antibiotic treatments [14].
Recent experimental and clinical studies suggest that the lack of efficient drugs against MDR, XDR, or PDR K. pneumoniae is not the only cause of treatment failures. Poor treatment outcomes may also result from the phenomenon of heteroresistance, which often leads to misdiagnoses and the inappropriate use of antibiotics.

2. Heteroresistance: Definition, Detection Methods, and Underlying Mechanisms

Antibiotic heteroresistance is a frequent phenotype observed in a wide range of pathogenic bacteria [15,16,17,18]. The terms “polyclonal” and “monoclonal” heteroresistance have been used to describe different forms of bacterial behavior in the presence of antibiotics [16]. In the case of polyclonal heteroresistance, genetically distinct clones exhibit stable resistant or susceptible phenotypes. Examples of polyclonal heteroresistance are co-infections by at least two isolates with different MIC (minimum inhibitory concentration) values or the emergence of stable resistant mutants during antibiotic treatment. In monoclonal heteroresistance, a subpopulation displays increased resistance transiently. Although this phenotype could be maintained for several generations, the resistant subpopulation often reverts to a susceptible phenotype. In the case of monoclonal heteroresistance, any resistant or susceptible cell may give rise to a new heteroresistant population [16,17]. The recently proposed definition of heteroresistance denotes the presence of a resistant subpopulation with an MIC at least eightfold higher than the highest concentration of the drug that does not affect the growth of the main population [16,18]. Other criteria that should be considered to classify clinical isolates as heteroresistant are the growth of the resistant subpopulation above the clinical breakpoint and its high frequency (above 1 × 10−7) [16].
Due to phenotypic and genetic instability, heteroresistance is difficult to detect and study [16,18,19]. Rapid molecular and biochemical methods used to identify specific antibiotic resistance, such as real-time PCR or VITEK 2, are insufficient to detect heteroresistance due to the lack of a genetic marker or low sensitivity. The most reliable and quantitative method for detecting heteroresistant strains is the population analysis profile (PAP) assay. To assess the frequency of resistant mutants, bacteria are spread on agar plates with increasing concentrations of antibiotics. A gradual decrease in the number of CFU instead of a single-step loss of survival indicates the presence of a resistant subpopulation (Figure 1a). Unfortunately, due to the duration and complexity of the PAP assay, this method is not feasible for clinical use, and heteroresistance often remains undetected [15,20]. Alternative methods to PAP are the disc diffusion assay and Etests (Figure 1a). In the case of heteroresistant isolates, resistant colonies appear within the clearing zone around the disk or Etest strips. Both methods’ limitations are that they are not quantitative and may often give false-positive or false-negative results [21,22].
Various advanced techniques have been adapted to evaluate heteroresistant bacteria in recent years. The correct identification of heteroresistance can be made possible by comparing the growth rates of many single cells at several antibiotic concentrations. Testing only a single colony of the primary bacteria population isolated from patients may lead to a misinterpretation and poor assessment of the sensitivity of the population [23]. Therefore, direct single-cell imaging combined with microfluidics could be a promising method for the rapid detection of heteroresistant cells, as shown by Baltekin et al. [24]. Whole-genome sequencing is an increasingly used method to detect resistant subpopulations in clinical isolates [25,26]. Bauer et al. developed an extended Raman-based antibiotic susceptibility test. This assay was based on monitoring the metabolic activity spectroscopically measured by the D2O uptake in two model bacteria, E. coli and Enterococcus faecalis [27]. To assess clarithromycin susceptibility in Helicobacter pylori, droplet digital PCR was applied using probes detecting the most common mutations of the 23S rRNA gene that cause resistance to clarithromycin [28].
It should be noted that heteroresistance is distinct from another form of population heterogeneity persistence, in which a small subpopulation of dormant cells is transiently resistant to antibiotics [29,30]. However, in contrast to heteroresistant populations, persisters are not able to replicate in the presence of antibiotics (Figure 1b). Although persisters are metabolically inactive, they may resume growth after the therapy causing recurrent infections.
The most common mechanisms underlying heteroresistance in Gram-negative bacteria are spontaneous tandem gene amplification [22,31,32,33]. For example, amplification of the prmD gene was observed in colistin-heteroresistant Salmonella typhimurium isolates [13]. PrmD is a positive regulator that induces the synthesis of proteins involved in the modification of lipid A (see next paragraph). The addition of an arabinose derivative and phosphoethanolamine decreases the net negative charge of lipid A, fortifies the outer membrane (OM), and leads to colistin resistance. Other studies have demonstrated that the unstable tobramycin resistance of Acinetobacter baumannii emerged due to the extensive RecA-dependent amplification of the aadB gene encoding an aminoglycoside adenylyltransferase [32]. Amplification of the lepB gene encoding the target of arylomicin, signal peptidase LepB, was recently detected in unstable arylomicin-heteroresistant E. coli subpopulations [33]. The number of lepB copies in different subpopulations was remarkably variable, from 1 to 50 copies. The upregulation of resistant genes may also result from mutations in the promoter regions. An insertion sequence (ISAba1) was found in the promoter region of a β-lactamase gene (blaADC) in imipenem-heteroresistant A. baumannii isolates [34]. Other mechanisms responsible for heteroresistance are the overproduction of efflux pumps and reduced expression of porins [35,36,37,38,39,40]. For example, heteroresistance to tigecycline in Salmonella enterica was associated with an increased level of the RamA transcriptional factor, resulting in the overexpression of the AcrAB-TolC and OqxAB efflux pumps [35]. Other studies have indicated that the downregulation of the OprD porin and overexpression of the efflux systems contributed to carbapenem and imipenem heteroresistance in Pseudomonas aeruginosa [38,41].
Mutations associated with heteroresistance often cause a high fitness cost [42]. Therefore, compensatory mutations are selected in the absence of antibiotics, and bacteria revert to a sensitive phenotype. These pseudorevertants have a genotype different from that of the susceptible ancestral isolate, even though they show a similar phenotype. Intrinsic instability of gene amplifications additionally contributes to the transient character of heteroresistance.
Heteroresistance may lead to antibiotic treatment failure due to the misclassification of heteroresistant isolates as susceptible strains. Consequently, inappropriate therapies fail to eradicate infections and facilitate the selection of resistant mutants [19,20,43,44]. It has been demonstrated that different antibiotics at levels below the MIC of the susceptible clinical isolates of E. coli and Salmonella enterica can cause the enrichment of resistant subpopulations with an increased copy number of resistance genes [19]. Band et al. reported that cumulative exposure of carbapenem-resistant Enterobacterales to β-lactams led to the development of heteroresistant isolates, which were gradually displaced by resistant cells [45]. Exposure to antibiotics and heteroresistance may also facilitate evasion of the host immune response. Napier et al. found that pretreatment of heteroresistant E. cloacae isolates with colistin resulted in cross-resistance to the host antimicrobial lysozyme [46]. In contrast, the colistin-sensitive E. cloacae isolates were not protected from lysozyme. Band et al. also revealed that the frequencies of resistant E. cloacae subpopulations in macrophage-depleted mice were decreased, indicating that the innate immune response may promote the appearance of resistant cells [47]. Indeed, it was found that specific innate host immune components (H2O2, lysozyme, the murine cationic antimicrobial peptide CRAMP, and its human orthologue LL37) increased the level of colistin-resistant subpopulations in vitro and in vivo.

3. Prevalence and Mechanisms of Heteroresistance in K. pneumoniae

Heteroresistance in K. pneumoniae has been described in several reports, as summarized in Table 1. Some of these studies have provided information about the molecular basis of emerging heteroresistant populations. It is worth noting that, apart from the core genome (~2000 genes), K. pneumoniae possesses a large set of accessory genes located in the chromosome or plasmids (~3000–4000 genes) and encoding specific virulence factors and antibiotic resistance mechanisms. Its high variability may contribute to population heterogeneity and favor the formation of heteroresistant subpopulations [3,46].

3.1. Heteroresistance to Colistin

The most commonly reported examples of K. pneumoniae heteroresistance with known mechanisms are clinical isolates that produce polymixin-resistant subpopulations (Table 1). Two closely related polymyxin antibiotics, colistin (polymyxin E) and polymyxin B., are used clinically. Colistin is a drug of last resort for infections caused by K. pneumoniae. In general, colistin kills Gram-negative pathogens, targeting the lipid A moiety of lipopolysaccharide (LPS) and destabilizing the OM by displacing divalent cations Ca2+ and Mg2+ that form bridges between LPS [66]. According to a recently proposed model, colistin also targets LPS molecules during its transport from the cytoplasm to the inner membrane (IM) and then to the OM. This colistin activity results in IM permeabilization, loss of cytoplasm content, cell lysis, and death [67]. Colistin resistance can appear via different mechanisms, most of which result in the addition of cationic moieties, 4-amino-4-deoxy-L-arabinose (L-Ara4N), and phosphoethanolamine (PEtN) to lipid A. Consequently, positively charged polymyxins are repelled from the membrane [66,67,68]. Lipid A hydroxylation and palmitoylation have recently been proposed as an alternative mechanism of colistin resistance [68]. The anionic capsular polysaccharide significantly limits the access of polymyxins to the OM. Hence, K. pneumoniae acapsular mutants show increased susceptibility to polymyxins [66].
In the first study that revealed mechanisms responsible for colistin heteroresistance in K. pneumoniae, Jayol et al. found that the colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP [50]. PhoP belongs to the PhoPQ two-component system, which is highly conserved among many Gram-negative bacteria (Figure 2). The sensor kinase PhoQ can respond by autophosphorylation to several environmental changes, including low pH, low Mg2+ concentrations, and the presence of antimicrobial peptides. The phosphate group is then transferred to the response regulator PhoP, which activates the expression of downstream genes involved in LPS modifications (Figure 2). The observed amino acid substitution in PhoP in colistin-resistant K. pneumoniae subpopulations caused the overexpression of the pmrHFIJKLM operon (also known as arnBCADTEF or pbgPE) responsible for L-Ara4N synthesis [50]. The PrmK transferase catalases the last step of the modification. The reversion of colistin resistance to susceptible phenotype was associated with the inactivation of PhoP caused by a partial deletion in the phoP gene.
Several studies have confirmed the contribution of the PhoPQ system in colistin heteroresistance [44,51,56]. It was proposed that an amino acid change in the sensor domain of PhoQ or PhoQ overexpression may lead to the constitutive phosphorylation of PhoP, and the subsequent activation of genes involved in lipid A modifications [51]. Colistin heteroresistance in K. pneumoniae was also attributed to mutations in the pmrAB genes, encoding another two-component system that controls the synthesis of L-Ara4N and PEtN (Figure 2) [21,56,57]. It is worth noting that diverse resistant subpopulations with different amino acid substitutions in PhoPQ and PmrAB were detected in various isolates. Evolution experiments have shown that unstable colistin-heteroresistance in K. pneumoniae might also emerge due to mutations in the crrB gene [55]. The CrrAB two-component system is involved in the control of the pmrHFIJKLM operon via the PmrAB regulatory network (Figure 2). Other studies have found that clinical crrB mutations lead to the addition of both L-Ara4N and pEtN to lipid A, inducing higher polymyxin resistance [69]. Colistin resistance may also be linked to mutations, insertions, or deletions in the mgrB, yciM, and lpxM genes [44,51,53,57]. The mgrB gene encodes a small transmembrane protein which acts as a negative regulator of the PhoPQ system. Therefore, its inactivation leads to overexpression of the pmrHFIJKLM operon [44,53,57]. The inactivation of the mgrB gene may also result in a thicker multilayered capsule which protects bacteria against colistin [70]. It is worth noting that inactivation of mgrB by point mutations, insertions, or deletions is one of the most common colistin resistance mechanisms in K. pneumoniae isolated from patients treated with colistin [63,71]. The role of yciM and lpxM genes in the emergence of colistin-resistant K. pneumoniae subpopulations is probably associated with the regulation of LPS biosynthesis and lipid acetylation [51].
Colistin heteroresistance may also be linked to the emergence of a small colony variant phenotype during K. pneumoniae biofilm formation [52]. It has been demonstrated that these slow-growing colistin-resistant bacteria were absent in a planktonic population that was colistin-susceptible. Biofilms are multicellular communities embedded in extracellular polymeric substances that may constitute a physical barrier for antibiotics and other stress factors [72,73,74]. The complex structure of the biofilm facilitates the formation of heterogeneous subpopulations with different gene expression patterns. The innermost layers of the biofilm have limited access to oxygen and nutrients, which, in turn, may inhibit metabolism or induce specific pathways that enable bacteria to survive under stressful conditions, including antibiotics.

3.2. Heteroresistance to Carbapenems

The expression of ESBLs, AmpC-β-lactamases (DHA, FOX), and carbapenemases is the main cause of β-lactams resistance in K. pneumoniae [3,75]. Plasmid-based ESBLs belong to the CTX-M, SHV, and TEM families and hydrolyze third-generation cephalosporins and aztreonam. The main K. pneumoniae carbapenemase (KPC) is the most common class A serine β-lactamase, encoded by the blaKPC gene carried on plasmids and transported in the Tn4401 transposon, which facilitates its efficient transfer between bacterial isolates [75]. The increasing number of new KPC variants encoded on mobile genetic elements and their dissemination in Gramm-negative bacteria has been recently reported [76,77]. In addition to KPC, K. pneumoniae may produce different metallo-β-lactamases, including New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-b-lactamase (VIM), imipenemase (IMP), and OXY-48-like carbapenemases [3,75]. The emergence of NDMs-producing K. pneumoniae is of great concern because such isolates exhibit multidrug resistance and spread rapidly. Multiple studies have demonstrated that the enhanced resistance of K. pneumoniae to β-lactams may also result from deletions or mutations within the major outer membrane porins OmpK35 and OmpK36. It was found that OmpK35 and OmpK36 produce larger and more permeable channels than their E. coli homologs OmpF and OmpC. When third-generation cephalosporins were introduced into clinical use, this difference explains why K. pneumoniae isolates were initially more sensitive to these antibiotics than E. coli strains [78]. Porin deficiency reduces the uptake of β-lactams and, in combination with ESBLs activity, may confer clinical resistance [40,62,79]. The emergence of imipenem-resistant subpopulations with decreased levels of OmpK36 was reported by Adams-Sapper et al. (Table 1). The imipenem MIC for the subpopulations increased at least 32-fold, providing that a carbapenemase encoded by the blaKPC-2 gene was expressed [58]. Individual isolates that lost OmpK36 upon imipenem exposure regained expression of the porin in drug-free media and reverted to the heteroresistant phenotype. However, most isolates lost OmpK36 permanently and maintained the high-level imipenem resistance. Pournaras et al. (2010) found that meropenem heteroresistance may result from the transient overexpression of the blaKPC-2 gene [61]. After daily passages on a meropenem-free medium, the resistant subpopulations reverted into meropenem susceptible strains.

3.3. Heteroresistance to Tetracyclines

Drug efflux and ribosomal protection are predominant mechanisms of tetracycline resistance in many Gram-negative bacteria [80]. To overcome this problem, new tetracycline derivatives—semisynthetic tigecycline and fully synthetic eravacycline—were developed [81]. In recent years, increasing numbers of tigecycline- and eravacycline-resistant K. pneumoniae isolates have been reported [82]. The resistant mechanisms include overproduction of the efflux pumps AcrAB, OqxAB, and MacAB. Expression of the acrAB and oqxAB genes is regulated by global transcriptional activators such as RamA, SoxS, and MarA, which are controlled by their transcriptional repressors RamR, SoxR, and MarR, respectively [39,82]. Tigecycline- and eravacycline-resistance is also caused by mutations in the ribosomal protein S10 (encoded by the rpsJ gene), which prevent the binding of the antibiotics to the small 30S ribosomal subunit [81].
So far, tigecycline- and eravacycline-heteroresistant isolates have been reported by three independent studies (Table 1) [39,63,64]. Multiple mechanisms have contributed to tigecycline resistance in heteroresistant subpopulations, including (1) the overexpression of the acrAB efflux pump genes; (2) mutations of the ramR gene, which resulted in the RamA upregulation and led to AcrAB-TolC efflux pump production; (3) mutation of the soxR gene, which resulted in the SoxS overexpression and AcrAB-TolC upregulation; (4) the nucleotide insertion and frameshifts in the rpsJ gene, which encodes the ribosomal S10 protein. These mutations might weaken tigecycline binding to 16S rRNA [63]. Other findings indicated that eravacycline heteroresistance might result from the overexpression of the OqxAB and MacAB efflux pumps and the transcriptional regulator RamA [39]. The heteroresistant eravacycline strains originated from the samples not pre-treated with tigecycline or erevacyclin. Therefore, the overexpression of AcrAB and OqzAB may indicate that the isolates were exposed to other compounds transported by these pumps.

3.4. Heteroresistance to Aminoglycosides

Aminoglycoside-modifying enzymes (AMEs) and the presence of 16S ribosomal RNA methyltransferases (armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA) are the main cause of aminoglycoside resistance in K. pneumoniae and other Enterobacterales [83,84]. AMEs include N-acetyltransferases, O-nucleotidyltransferases, and O-phosphotransferases, which covalently modify specific amino or hydroxyl moieties on the aminoglycosides.
Aminoglycoside heteroresistance in K. pneumoniae isolates was mainly attributed to the amplification of the AMEs genes that were harbored on plasmids and encoded aminoglycosides acetyl-transferases (aac) (Table 1) [22]. In the presence of gentamycin, tobramycin, or netilmicin, the number of plasmid copies and aac copies per plasmid increased. The resistance phenotype was unstable, and after 40 generations without the pressure of antibiotics, the number of aac copies and plasmid copies decreased, or the plasmid was lost. Consequently, the MIC values reverted to that of the parental isolates. Amikacin-resistant subpopulations had mutations in the ubiJ and cydA genes, which are associated with a small colony variant phenotype or aminoglycoside resistance. During growth in the absence of amikacin, compensatory point mutations or duplications (ubiJ) were selected, leading to amikacin susceptibility of the revertants. The reversion to aminoglycoside susceptibility in the absence of selective pressure resulted from the fitness cost conferred by the acc gene amplification and amikacin resistance mutations [22]. It has also been found that amikacin heteroresistance can be associated with the unstable overexpression of aac genes and several mutations (SNVs, single nucleotide variations) in genes encoding proteins involved in various physiological processes, including carbohydrate uptake and chromosome segregation [60].

4. Therapeutic Strategies against Heteroresistant K. pneumoniae

Recent findings suggest that combinations of clinically approved antibiotics might eradicate infections associated with heteroresistant K. pneumoniae (Table 2) [56,64,85,86]. Cheong et al. found that meropenem combined with colistin might be a therapeutic option for infections caused by colistin-heteroresistant K. pneumoniae [56]. Meropenem alone eradicated a heteroresistant isolate, but its combination with colistin was more effective. Tian et al. investigated the combined effect of polymyxin B and tigecycline against K. pneumoniae isolates manifesting dual heteroresistance to both antibiotics [64]. As expected, antibiotic monotherapy (polymyxin B or tigecycline) had a transient inhibitory effect. Susceptible bacteria were killed, but resistant subpopulations were not eliminated. The combination of polymyxin B and tigecycline, used at reduced doses, successfully eradicated susceptible and resistant subpopulations. In other studies, the combination of ceftazidime/avibactam (β-lactam/β-lactamase inhibitor combination) improved the efficacy of polymyxin B against heteroresistant carbapenemase-producing K. pneumoniae isolates and hindered the emergence of polymyxin-resistant subpopulations in vitro [86]. Band et al. investigated different combinations of antibiotics against a K. pneumoniae isolate that was heteroresistant to colistin and fosfomycin [85]. Colistin and fosfomycin monotherapy did not inhibit infection in mice, but the combination of both drugs significantly reduced the level of bacteria. Another tested isolate exhibited homogenous resistance to colistin and heteroresistance to fosfomycin and ceftazidime. Only dual therapy with fosfomycin and ceftazidime eradicated the infection, while any combination with colistin (colistin + fosfomycin or colistin + ceftazidime) was not effective. These results indicate that multidrug therapies should use antibiotics targeting different heteroresistant subpopulations rather than antibiotics targeting homogeneously resistant populations. Further studies revealed that this approach resulted in decreased survival, even in the case of two pandrug-resistant K. pneumoniae isolates known to cause untreatable infections (Table 2) [85].

5. Concluding Remarks

Numerous recent studies have revealed that heteroresistance occurs relatively often in K. pneumoniae clinical isolates (Table 1). Mutations and mechanisms responsible for the emergence of heteroresistant K. pneumoniae subpopulations have been previously identified as antibiotic-resistant factors in Klebsiella and other Enterobacteriaceae. These mechanisms have often contributed to unstable heteroresistance and have included lipid A modifications, the overexpression of pump systems, or gene amplification.
Although it has been well documented that bacterial biofilms are highly heterogeneous populations that are more drug-resistant than planktonic bacteria, little is known about the influence of biofilm’s environment on heteroresistance. The presence of colistin-heteroresistant subpopulations in K. pneumoniae biofilms is the only example described so far [52,73]. It was found that planktonic cells were susceptible to colistin, whereas colistin resistance exclusively detected within the biofilm population was linked with the small colony variant phenotype. Contrary to these results, Zhang et al. demonstrated that slow growth and biofilm formation do not contribute to amikacin heteroresistance in K. pneumoniae [60].
It should be kept in mind that most experiments focused on heteroresistance have been performed in vitro or using animal models. Therefore, these results may not reflect the mechanisms occurring in the host organism. Many additional issues should be considered, e.g., the interplay between the pathogen and the host immune response, as well as the co-existence of several heterogeneous subpopulations, including persister and VBNC (viable but non-culturable) bacteria, which are difficult to detect [17]. The impact of heteroresistance on antibiotic treatment failure has been a matter of extensive debate. However, increasing data strongly suggest that heteroresistance may be responsible for poor outcomes and recurrent infections with pathogens classified as susceptible. The mathematical modeling of pharmacodynamics of heteroresistance in K. pneumoniae and other species supports this assumption [22]. Recurrent heteroresistant infections treated repeatedly with the same antibiotic may allow the evolution of drug-resistant bacteria [60]. Therefore, it is crucial to improve methods for detecting heteroresistance and develop effective therapeutic strategies, which should be based on a combination of antibiotics as suggested by recent studies.
It has been recently demonstrated that human contact restrictions and other strategies to prevent SARS-CoV-2 transmission during the COVID-19 pandemic have limited the spread of K. pneumoniae and other ESKAPE pathogens [87,88]. However, the extensive use of disinfectants and increased administration of antibiotics to prevent bacterial co-infections during the COVID-19 pandemic may enhance the emergence and spread of drug resistance [89,90,91]. Several studies have indicated that the prevalence of carbapenem-resistant K. pneumoniae co-infection in COVID-19 patients may reach more than 50% of cases [92,93]. Moreover, K. pneumoniae heteroresistance toward chlorhexidine, the most widely used disinfectant in hospitals, has been previously described (Table 1) [65]. Given the lack of standard procedures and the difficulties in detecting heteroresistance, further studies are urgently needed to understand the mechanisms underlying K. pneumoniae hetero resistance and prevent the spread of multidrug-resistant infections.

Author Contributions

Conceptualization, K.S.-S., D.K.-W. and E.L.; writing—original draft preparation, K.S.-S., A.Ł., D.K.-W. and E.L.; writing—review and editing, K.S.-S., A.Ł., D.K.-W. and E.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the University of Gdansk (grant No. 531/D010-D241-21).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AMEsAminoglycoside modifying enzymes
cKPClassical Klebsiella pneumoniae
ESBLExtended-spectrum beta-lactamases
hvKPHypervirulent Klebsiella pneumoniae
IMInner membrane
L-Ara4N4-amino-4-deoxy-L-arabinose
MDRMultidrug-resistant
MICMinimum inhibitory concentration
OMOuter membrane
PAPPopulation analysis profile
PEtNPhosphoethanolamine
PDRPandrug-resistant
SNVsSingle nucleotide variations
XDRExtensively drug-resistant

References

  1. Paczosa, M.K.; Mecsas, J. Klebsiella pneumoniae: Going on the offense with a strong defense. Microbiol. Mol. Biol. Rev. 2016, 80, 629–661. [Google Scholar] [CrossRef] [Green Version]
  2. Piperaki, E.T.; Syrogiannopoulos, G.A.; Tzouvelekis, L.S.; Daikos, G.L. Klebsiella pneumoniae: Virulence, biofilm and antimicrobial resistance. Pediatr. Infect. Dis. J. 2017, 36, 1002–1005. [Google Scholar] [CrossRef] [PubMed]
  3. Martin, R.M.; Bachman, M.A. Colonization, infection, and the accessory genome of Klebsiella pneumoniae. Front. Cell. Infect. Microbiol. 2018, 8, 4. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Russo, T.A.; Marr, C.M. Hypervirulent Klebsiella pneumoniae. Clin. Microbiol. Rev. 2019, 32, e00001-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Navon-Venezia, S.; Kondratyeva, K.; Carattoli, A. Klebsiella pneumoniae: A major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol. Rev. 2017, 41, 252–275. [Google Scholar] [CrossRef]
  6. Xu, J.; Zhao, Z.; Ge, Y.; He, F. Rapid emergence of a pandrug-resistant Klebsiella pneumoniae ST11 isolate in an inpatient in a teaching hospital in China after treatment with multiple broad-spectrum antibiotics. Infect. Drug Resist. 2020, 13, 799–804. [Google Scholar] [CrossRef] [Green Version]
  7. Zowawi, H.M.; Forde, B.M.; Alfaresi, M.; Alzarouni, A.; Farahat, Y.; Chong, T.M.; Yin, W.F.; Chan, K.G.; Li, J.; Schembri, M.A.; et al. Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae. Sci. Rep. 2015, 5, 15082. [Google Scholar] [CrossRef]
  8. Prokesch, B.C.; TeKippe, M.; Kim, J.; Raj, P.; TeKippe, E.M.E.; Greenberg, D.E. Primary osteomyelitis caused by hypervirulent Klebsiella pneumoniae. Lancet Infect. Dis. 2016, 16, e190–e195. [Google Scholar] [CrossRef]
  9. Patel, P.K.; Russo, T.A.; Karchmer, A.W. Hypervirulent Klebsiella pneumoniae. Open Forum Infect. Dis. 2014, 1, ofu028. [Google Scholar] [CrossRef] [Green Version]
  10. Struve, C.; Roe, C.C.; Stegger, M.; Stahlhut, S.G.; Hansen, D.S.; Engelthaler, D.M.; Andersen, P.S.; Driebe, E.M.; Keim, P.; Krogfelt, K.A. Mapping the evolution of hypervirulent Klebsiella pneumoniae. mBio 2015, 6, e00630-15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  11. Shon, A.S.; Bajwa, R.P.S.; Russo, T.A. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: A new and dangerous breed. Virulence 2013, 4, 107–118. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Zhang, Y.; Zeng, J.; Liu, W.; Zhao, F.; Hu, Z.; Zhao, C.; Wang, Q.; Wang, X.; Chen, H.; Li, H.; et al. Emergence of a hypervirulent carbapenem-resistant Klebsiella pneumoniae isolate from clinical infections in China. J. Infect. 2015, 71, 553–560. [Google Scholar] [CrossRef] [PubMed]
  13. Li, P.; Liang, Q.; Liu, W.; Zheng, B.; Liu, L.; Wang, W.; Xu, Z.; Huang, M.; Feng, Y. Convergence of carbapenem resistance and hypervirulence in a highly-transmissible ST11 clone of K. pneumoniae: An epidemiological, genomic and functional study. Virulence 2021, 12, 377–388. [Google Scholar] [CrossRef] [PubMed]
  14. Mulani, M.S.; Kamble, E.E.; Kumkar, S.N.; Tawre, M.S.; Pardesi, K.R. Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: A review. Front. Microbiol. 2019, 10, 539. [Google Scholar] [CrossRef]
  15. Band, V.I.; Weiss, D.S. Heteroresistance: A cause of unexplained antibiotic treatment failure? PLoS Pathog. 2019, 15, e1007726. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Andersson, D.I.; Nicoloff, H.; Hjort, K. Mechanisms and clinical relevance of bacterial heteroresistance. Nat. Rev. Microbiol. 2019, 17, 479–496. [Google Scholar] [CrossRef]
  17. Dewachter, L.; Fauvart, M.; Michiels, J. Bacterial heterogeneity and antibiotic survival: Understanding and combatting persistence and heteroresistance. Mol. Cell 2019, 76, 255–267. [Google Scholar] [CrossRef]
  18. El-Halfawy, O.M.; Valvano, M.A. Antimicrobial heteroresistance: An emerging field in need of clarity. Clin. Microbiol. Rev. 2015, 28, 191–207. [Google Scholar] [CrossRef] [Green Version]
  19. Pereira, C.; Larsson, J.; Hjort, K.; Elf, J.; Andersson, D.I. The highly dynamic nature of bacterial heteroresistance impairs its clinical detection. Commun. Biol. 2021, 4, 521. [Google Scholar] [CrossRef] [PubMed]
  20. Choby, J.E.; Ozturk, T.; Satola, S.W.; Jacob, J.T.; Weiss, D.S. Widespread cefiderocol heteroresistance in carbapenem-resistant gram-negative pathogens. Lancet Infect. Dis. 2021, 21, 597–598. [Google Scholar] [CrossRef]
  21. Seo, J.; Wi, Y.M.; Kim, J.M.; Kim, Y.J.; Ko, K.S. Detection of colistin-resistant populations prior to antibiotic exposure in kpc-2-producing Klebsiella pneumoniae clinical isolates. J. Microbiol. 2021, 59, 590–597. [Google Scholar] [CrossRef] [PubMed]
  22. Nicoloff, H.; Hjort, K.; Levin, B.R.; Andersson, D.I. The High Prevalence of Antibiotic Heteroresistance in Pathogenic Bacteria Is Mainly Caused by Gene Amplification. Nat. Microbiol. 2019, 4, 504–514. [Google Scholar] [CrossRef]
  23. Balaji, V.; Jeremiah, S.S.; Baliga, P.R. Polymyxins: Antimicrobial susceptibility concerns and therapeutic options. Indian J. Med. Microbiol. 2011, 29, 230–242. [Google Scholar] [CrossRef]
  24. Baltekin, Ö.; Boucharin, A.; Tano, E.; Andersson, D.I.; Elf, J. Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging. Proc. Natl. Acad. Sci. USA 2017, 114, 9170–9175. [Google Scholar] [CrossRef] [Green Version]
  25. Bradley, P.; Gordon, N.C.; Walker, T.M.; Dunn, L.; Heys, S.; Huang, B.; Earle, S.; Pankhurst, L.J.; Anson, L.; de Cesare, M.; et al. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis. Nat. Commun. 2015, 6, 10063. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Operario, D.J.; Koeppel, A.F.; Turner, S.D.; Bao, Y.; Pholwat, S.; Banu, S.; Foongladda, S.; Mpagama, S.; Gratz, J.; Ogarkov, O.; et al. Prevalence and extent of heteroresistance by next generation sequencing of multidrug-resistant tuberculosis. PLoS ONE 2017, 12, e0176522. [Google Scholar]
  27. Bauer, D.; Wieland, K.; Qiu, L.; Neumann-Cip, A.C.; Magistro, G.; Stief, C.; Wieser, A.; Haisch, C. Heteroresistant bacteria detected by an extended Raman-based antibiotic susceptibility test. Anal. Chem. 2020, 92, 8722–8731. [Google Scholar] [CrossRef] [PubMed]
  28. Sun, L.; Talarico, S.; Yao, L.; He, L.; Self, S.; You, Y.; Zhang, H.; Zhang, Y.; Guo, Y.; Liu, G.; et al. Droplet dDigital PCR-based detection of clarithromycin resistance in Helicobacter pylori isolates reveals frequent heteroresistance. J. Clin. Microbiol. 2018, 56, e00019-18. [Google Scholar] [CrossRef] [Green Version]
  29. Gollan, B.; Grabe, G.; Michaux, C.; Helaine, S. Bacterial persisters and infection: Past, present, and progressing. Ann. Rev. Microbiol. 2019, 73, 359–385. [Google Scholar] [CrossRef]
  30. Balaban, N.Q.; Helaine, S.; Lewis, K.; Ackermann, M.; Aldridge, B.; Andersson, D.I.; Brynildsen, M.P.; Bumann, D.; Camilli, A.; Collins, J.J.; et al. Definitions and guidelines for research on antibiotic persistence. Nat. Rev. Microbiol. 2019, 17, 441–448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  31. Hjort, K.; Nicoloff, H.; Andersson, D.I. Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica. Mol. Microbiol. 2016, 102, 274–289. [Google Scholar] [CrossRef]
  32. Anderson, S. E Aminoglycoside heteroresistance in Acinetobacter baumannii AB5075. mSphere 2018, 3, e00271-18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Girgis, H.S.; Dupai, C.D.; Lund, J.; Reeder, J.; Guillory, J.; Durinck, S.; Liang, Y.; Kaminker, J.; Smith, P.A.; Skippington, E. Single-molecule nanopore sequencing reveals extremetarget copy number heterogeneity inarylomycin-resistant mutants. Proc. Natl. Acad. Sci. USA 2021, 118, e2021958118. [Google Scholar] [CrossRef] [PubMed]
  34. Lee, Y.; Kim, C.K.; Lee, H.; Jeong, S.H.; Yong, D.; Lee, K. A novel insertion sequence, ISAba10, inserted into ISAba1 adjacent to the BlaOXA-23 gene and disrupting the outer membrane protein gene carO in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2011, 55, 361–363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Chen, Y.; Hu, D.; Zhang, Q.; Liao, X.P.; Liu, Y.H.; Sun, J. Efflux pump overexpression contributes to tigecycline heteroresistance in Salmonella enterica serovar typhimurium. Front. Cell. Infect. Microbiol. 2017, 7, 37. [Google Scholar] [CrossRef] [PubMed]
  36. Karakonstantis, S.; Saridakis, I. Colistin heteroresistance in Acinetobacter spp.: Systematic review and meta-analysis of the prevalence and discussion of the mechanisms and potential therapeutic implications. Int. J. Antimicrob. Agents 2020, 56, 106065. [Google Scholar] [CrossRef]
  37. Kang, K.N.; Klein, D.R.; Kazi, M.I.; Guérin, F.; Cattoir, V.; Brodbelt, J.S.; Boll, J.M. Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ-dependent 4-amino-4-deoxy-l-arabinose addition to lipid A. Mol. Microbiol. 2019, 111, 1604–1616. [Google Scholar] [CrossRef] [PubMed]
  38. Ikonomidis, A.; Tsakris, A.; Kantzanou, M.; Spanakis, N.; Maniatis, A.N.; Pournaras, S. Efflux system overexpression and decreased OprD contribute to the carbapenem heterogeneity in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 2008, 279, 36–39. [Google Scholar] [CrossRef] [Green Version]
  39. Zheng, J.; Lin, Z.; Sun, X.; Lin, W.; Chen, Z.; Wu, Y.; Qi, G.; Deng, Q.; Qu, D.; Yu, Z. Overexpression of OqxAB and MacAB efflux pumps contributes to eravacycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae. Emerg. Microbes Infect. 2018, 7, 1–11. [Google Scholar] [CrossRef] [Green Version]
  40. Mmatli, M.; Mbelle, N.M.; Maningi, N.E.; Osei Sekyere, J. Emerging transcriptional and genomic mechanisms mediating carbapenem and polymyxin resistance in Enterobacteriaceae: A systematic review of current reports. mSystems 2020, 5, e00783-20. [Google Scholar] [CrossRef]
  41. Xu, Y.; Zheng, X.; Zeng, W.; Chen, T.; Liao, W.; Qian, J.; Lin, J.; Zhou, C.; Tian, X.; Cao, J.; et al. Mechanisms of heteroresistance and resistance to imipenem in Pseudomonas aeruginosa. Infect. Drug Resist. 2020, 13, 1419–1428. [Google Scholar] [CrossRef]
  42. Vogwill, T.; Maclean, R.C. The genetic basis of the fitness costs of antimicrobial resistance: A meta-analysis approach. Evol. Appl. 2015, 8, 284–295. [Google Scholar] [CrossRef]
  43. Band, V.I.; Satola, S.W.; Smith, R.D.; Hufnagel, D.A.; Bower, C.; Conley, A.B.; Rishishwar, L.; Dale, S.E.; Hardy, D.J.; Vargas, R.L.; et al. Colistin heteroresistance is largely undetected among carbapenem-resistant Enterobacterales in the United States. mBio 2021, 12, e02881-20. [Google Scholar] [CrossRef]
  44. Band, V.I.; Satola, S.W.; Burd, E.M.; Farley, M.M.; Jacob, J.T.; Weiss, D.S. Carbapenem-resistant Klebsiella pneumoniae exhibiting clinically undetected colistin heteroresistance leads to treatment failure in a murine model of infection. mBio 2018, 9, e02448-17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Band, V.I.; Weiss, D.S. Heteroresistance to beta-lactam antibiotics may often be a stage in the progression to antibiotic resistance. PLoS Biol. 2021, 19, e3001346. [Google Scholar] [CrossRef] [PubMed]
  46. Napier, B.A.; Band, V.; Burd, E.M.; Weiss, D.S. Colistin heteroresistance in Enterobacter cloacae is associated with cross-resistance to the host antimicrobial lysozyme. Antimicrob. Agents Chemother. 2014, 58, 5594–5597. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Band, V.I.; Crispell, E.K.; Napier, B.A.; Herrera, C.M.; Tharp, G.K.; Vavikolanu, K.; Pohl, J.; Read, T.D.; Bosinger, S.E.; Trent, M.S.; et al. Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae. Nat. Microbiol. 2016, 1, 16053. [Google Scholar] [CrossRef] [PubMed]
  48. Poudyal, A.; Howden, B.P.; Bell, J.M.; Gao, W.; Owen, R.J.; Turnidge, J.D.; Nation, R.L.; Li, J. In vitro pharmacodynamics of colistin against multidrug-resistant Klebsiella pneumoniae. J. Antimicrob. Chemother. 2008, 62, 1311–1318. [Google Scholar] [CrossRef] [PubMed]
  49. Meletis, G.; Tzampaz, E.; Sianou, E.; Tzavaras, I.; Sofianou, D. Colistin heteroresistance in carbapenemase-producing Klebsiella pneumoniae. J. Antimicrob. Chemother. 2011, 66, 946–947. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Jayol, A.; Nordmann, P.; Brink, A.; Poirel, L. Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system. Antimicrob. Agents Chemother. 2015, 59, 2780–2784. [Google Scholar] [CrossRef] [Green Version]
  51. Halaby, T.; Kucukkose, E.; Janssen, A.B.; Rogers, M.R.C.; Doorduijn, D.J.; van der Zanden, A.G.M.; al Naiemi, N.; Vandenbroucke-Grauls, C.M.J.E.; van Schaik, W. Genomic characterization of colistin heteroresistance in Klebsiella pneumoniae during a nosocomial outbreak. Antimicrob. Agents Chemother. 2016, 60, 6837–6843. [Google Scholar] [CrossRef] [Green Version]
  52. Silva, A.; Sousa, A.M.; Alves, D.; Lourenço, A.; Pereira, M.O. Heteroresistance to colistin in Klebsiella pneumoniae is triggered by small colony variants sub-populations within biofilms. Pathog. Dis. 2016, 74, ftw036. [Google Scholar] [CrossRef] [Green Version]
  53. Bardet, L.; Baron, S.; Leangapichart, T.; Okdah, L.; Diene, S.M.; Rolain, J.-M. Deciphering heteroresistance to colistin in a Klebsiella pneumoniae isolate from Marseille, France. Antimicrob. Agents Chemother. 2017, 61, e00356-17. [Google Scholar] [CrossRef] [Green Version]
  54. Juhász, E.; Iván, M.; Pintér, E.; Pongrácz, J.; Kristóf, K. Colistin resistance among blood culture isolates at a tertiary care centre in Hungary. J. Glob. Antimicrob. Resist. 2017, 11, 167–170. [Google Scholar] [CrossRef]
  55. Cain, A.K.; Boinett, C.J.; Barquist, L.; Dordel, J.; Fookes, M.; Mayho, M.; Ellington, M.J.; Goulding, D.; Pickard, D.; Wick, R.R.; et al. Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin. Sci. Rep. 2018, 8, 9868. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Cheong, H.S.; Kim, S.Y.; Wi, Y.M.; Peck, K.R.; Ko, K.S. Colistin heteroresistance in Klebsiella pneumoniae isolates and diverse mutations of PmrAB and PhoPQ in resistant subpopulations. J. Clin. Med. 2019, 8, 1444. [Google Scholar] [CrossRef] [Green Version]
  57. Morales-León, F.; Lima, C.A.; González-Rocha, G.; Opazo-Capurro, A.; Bello-Toledo, H. Colistin Heteroresistance among extended spectrum β-lactamases-producing Klebsiella pneumoniae. Microorganisms 2020, 8, 1279. [Google Scholar] [CrossRef] [PubMed]
  58. Adams-Sapper, S.; Nolen, S.; Donzelli, G.F.; Lal, M.; Chen, K.; da Silva, L.H.J.; Moreira, B.M.; Riley, L.W. Rapid induction of high-level carbapenem resistance in heteroresistant KPC-producing Klebsiella pneumoniae. Antimicrob. Agents Chemother. 2015, 59, 3281–3289. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Tato, M.; Morosini, M.; García, L.; Albertí, S.; Coque, M.T.; Cantón, R. Carbapenem heteroresistance in VIM-1-producing Klebsiella pneumoniae isolates belonging to the same clone: Consequences for routine susceptibility testing. J. Clin. Microb. 2010, 48, 4089–4093. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Zhang, F.; Li, Q.; Bai, J.; Ding, M.; Yan, X.; Wang, G.; Zhu, B.; Zhou, Y. Heteroresistance to amikacin in carbapenem-resistant Klebsiella pneumoniae Strains. Front. Microbiol. 2021, 12, 3502. [Google Scholar] [CrossRef]
  61. Pournaras, S.; Kristo, I.; Vrioni, G.; Ikonomidis, A.; Poulou, A.; Petropoulou, D.; Tsakris, A. Characteristics of meropenem heteroresistance in Klebsiella pneumoniae carbapenemase (KPC)-producing clinical isolates of K. pneumoniae. J. Clin. Microb. 2010, 48, 2601–2604. [Google Scholar] [CrossRef] [Green Version]
  62. López-Camacho, E.; Paño-Pardo, J.R.; Sotillo, A.; Elías-López, C.; Martínez-Martínez, L.; Gómez-Gil, R.; Mingorance, J. Meropenem heteroresistance in clinical isolates of OXA-48–producing Klebsiella pneumoniae. Diagn. Microbiol. Infect. Dis. 2019, 93, 162–166. [Google Scholar] [CrossRef] [Green Version]
  63. Zhang, Q.; Lin, L.; Pan, Y.; Chen, J. Characterization of tigecycline-heteroresistant Klebsiella pneumoniae clinical isolates from a Chinese tertiary care teaching hospital. Front. Microbiol. 2021, 12, 2178. [Google Scholar] [CrossRef]
  64. Tian, Y.; Zhang, Q.; Wen, L.; Chen, J. Combined effect of polymyxin B and tigecycline to overcome heteroresistance in carbapenem-resistant Klebsiella pneumoniae. Microbiol. Spectr. 2021, 9, e0015221. [Google Scholar] [CrossRef]
  65. Naparstek, L.; Carmeli, Y.; Chmelnitsky, I.; Banin, E.; Navon-Venezia, S. Reduced susceptibility to chlorhexidine among extremely-drug-resistant strains of Klebsiella pneumoniae. J. Hosp. Infect. 2012, 81, 15–19. [Google Scholar] [CrossRef]
  66. Moffatt, J.H.; Harper, M.; Boyce, J.D. Mechanisms of polymyxin resistance. In Advances in Experimental Medicine and Biology; Springer: New York, NY, USA, 2019; Volume 1145, pp. 55–71. [Google Scholar]
  67. Sabnis, A.; Hagart, K.L.H.; Klöckner, A.; Becce, M.; Evans, L.E.; Furniss, R.C.D.; Mavridou, D.A.I.; Murphy, R.; Stevens, M.M.; Davies, J.C.; et al. Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. eLife 2021, 10, e65836. [Google Scholar] [CrossRef]
  68. Elias, R.; Duarte, A.; Perdigão, J. A molecular perspective on colistin and Klebsiella pneumoniae: Mode of action, resistance genetics, and phenotypic susceptibility. Diagnostics 2021, 11, 1165. [Google Scholar] [CrossRef] [PubMed]
  69. McConville, T.H.; Annavajhala, M.K.; Giddins, M.J.; Macesic, N.; Herrera, C.M.; Rozenberg, F.D.; Bhushan, G.L.; Ahn, D.; Mancia, F.; Trent, M.S.; et al. CrrB positively regulates high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Cell Rep. 2020, 3, 108313. [Google Scholar] [CrossRef] [PubMed]
  70. Formosa, C.; Herold, M.; Vidaillac, C.; Duval, R.E.; Dague, E. Unravelling of a mechanism of resistance to colistin in Klebsiella pneumoniae using atomic force microscopy. J. Antimicrob. Chemother. 2015, 70, 2261–2270. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Berglund, B. Acquired resistance to colistin via chromosomal and plasmid-mediated mechanisms in Klebsiella pneumoniae. Infect. Microb. Dis. 2019, 1, 10–19. [Google Scholar] [CrossRef]
  72. Hall, C.W.; Mah, T.F. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol. Rev. 2017, 41, 276–301. [Google Scholar] [CrossRef]
  73. Lopes, S.P.; Jorge, P.; Sousa, A.M.; Pereira, M.O. Discerning the role of polymicrobial biofilms in the ascent, prevalence, and extent of heteroresistance in clinical practice. Crit. Rev. Microbiol. 2021, 47, 162–191. [Google Scholar] [CrossRef]
  74. Van Acker, H.; van Dijck, P.; Coenye, T. Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends Microbiol. 2014, 22, 326–333. [Google Scholar] [CrossRef]
  75. Spagnolo, A.M.; Orlando, P.; Panatto, D.; Perdelli, F.; Cristina, M.L. An overview of carbapenem-resistant Klebsiella pneumoniae: Epidemiology and control measures. Rev. Med. Microbiol. 2014, 25, 7–14. [Google Scholar] [CrossRef]
  76. Tooke, C.L.; Hinchliffe, P.; Bonomo, R.A.; Schofield, C.J.; Mulholland, A.J.; Spencer, J. Natural variants modify Klebsiella pneumonia carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. J. Biol. Chem. 2021, 296, 100126. [Google Scholar] [CrossRef]
  77. Li, X.; Quan, J.; Ke, H.; Wu, W.; Feng, Y.; Yu, Y.; Jiang, Y. Emergence of a KPC variant conferring resistance to ceftazidime-avibactam in a widespread ST11 carbapenem-resistant Klebsiella pneumoniae clone in China. Front. Microbiol. 2021, 12, 2419. [Google Scholar] [CrossRef]
  78. Sugawara, E.; Kojima, S.; Nikaido, H. Klebsiella pneumoniae major porins OmpK35 and OmpK36 allow more efficient diffusion of β-lactams than their Escherichia coli homologs OmpF and OmpC. J. Bacteriol. 2016, 198, 3200–3208. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Wyres, K.L.; Lam, M.M.C.; Holt, K.E. Population genomics of Klebsiella pneumoniae. Nat. Rev. Microbiol. 2020, 18, 344–359. [Google Scholar] [CrossRef] [PubMed]
  80. Ni, R.T.; Onishi, M.; Mizusawa, M.; Kitagawa, R.; Kishino, T.; Matsubara, F.; Tsuchiya, T.; Kuroda, T.; Ogawa, W. The Role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae. Sci. Rep. 2020, 10, 81877. [Google Scholar] [CrossRef] [PubMed]
  81. Grossman, T.H. Tetracycline antibiotics and resistance. Cold Spring Harb. Perspect. Med. 2016, 6, a025387. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Bialek-Davenet, S.; Lavigne, J.P.; Guyot, K.; Mayer, N.; Tournebize, R.; Brisse, S.; Leflon-Guibout, V.; Nicolas-Chanoine, M.H. Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae. J. Antimicrob. Chemother. 2015, 70, 81–88. [Google Scholar] [CrossRef] [Green Version]
  83. Liang, C.; Xing, B.; Yang, X.; Fu, Y.; Feng, Y.; Zhang, Y. Molecular epidemiology of aminoglycosides resistance on Klebsiella pneumonia in a hospital in China. Int. J. Clin. Exp. Med. 2015, 8, 1381–1385. [Google Scholar]
  84. Foudraine, D.E.; Strepis, N.; Stingl, C.; ten Kate, M.T.; Verbon, A.; Klaassen, C.H.W.; Goessens, W.H.F.; Luider, T.M.; Dekker, L.J.M. Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics. Sci. Rep. 2021, 11, 12472. [Google Scholar] [CrossRef] [PubMed]
  85. Band, V.I.; Hufnagel, D.A.; Jaggavarapu, S.; Sherman, E.X.; Wozniak, J.E.; Satola, S.W.; Farley, M.M.; Jacob, J.T.; Burd, E.M.; Weiss, D.S. Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection. Nat. Microbiol. 2019, 4, 1627–1635. [Google Scholar] [CrossRef] [PubMed]
  86. Ma, X.; He, Y.; Yu, X.; Cai, Y.; Zeng, J.; Cai, R.; Lu, Y.; Chen, L.; Chen, C.; Huang, B. Ceftazidime/avibactam improves the antibacterial efficacy of polymyxin B against polymyxin B heteroresistant KPC-2-producing Klebsiella pneumoniae and hinders emergence of resistant subpopulation in vitro. Front. Microbiol. 2019, 10, 2029. [Google Scholar] [CrossRef]
  87. Gaspari, R.; Spinazzola, G.; Teofili, L.; Avolio, A.W.; Fiori, B.; Maresca, G.M.; Spanu, T.; Nicolotti, N.; de Pascale, G.; Antonelli, M. Protective effect of SARS-CoV-2 preventive measures against ESKAPE and Escherichia coli infections. Eur. J. Clin. Investig. 2021, 51, e13687. [Google Scholar] [CrossRef]
  88. Micozzi, A.; Assanto, G.M.; Cesini, L.; Minotti, C.; Cartoni, C.; Capria, S.; Ciotti, G.; Fegatelli, D.A.; Donzelli, L.; Martelli, M.; et al. Reduced transmission of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) in patients with haematological malignancies hospitalized in an Italian hospital during the COVID-19 pandemic. JAC-Antimicrob. Resist. 2021, 3, dlab167. [Google Scholar] [CrossRef] [PubMed]
  89. Lobie, T.A.; Roba, A.A.; Booth, J.A.; Kristiansen, K.I.; Aseffa, A.; Skarstad, K.; Bjørås, M. Antimicrobial resistance: A challenge awaiting the post-COVID-19 era. Int. J. Infect. Dis. 2021, 111, 322–325. [Google Scholar] [CrossRef] [PubMed]
  90. Wand, M.E.; Bock, L.J.; Bonney, L.C.; Sutton, J.M. Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine. Antimicrob. Agents Chemother. 2017, 61, e01162-16. [Google Scholar] [CrossRef] [Green Version]
  91. O’Toole, R.F. The interface between COVID-19 and bacterial healthcare-associated infections. Clin. Microbiol. Infect. 2021, 27, 1772–1776. [Google Scholar] [CrossRef]
  92. Mędrzycka-Dabrowska, W.; Lange, S.; Zorena, K.; Dabrowski, S.; Ozga, D.; Tomaszek, L. Carbapenem-resistant Klebsiella pneumoniae infections in ICU COVID-19 Patients—A scoping review. J. Clin. Med. 2021, 10, 2067. [Google Scholar] [CrossRef] [PubMed]
  93. Montrucchio, G.; Corcione, S.; Sales, G.; Curtoni, A.; de Rosa, F.G.; Brazzi, L. Carbapenem-resistant Klebsiella pneumoniae in ICU-admitted COVID-19 patients: Keep an eye on the ball. J. Glob. Antimicrob. Resis. 2020, 23, 398–400. [Google Scholar] [CrossRef] [PubMed]
Ijms 23 00449 g001 550
Figure 1. Heteroresistance detection methods and the difference between persister, resistant, and heteroresistant bacteria. (a) In the PAP assay, bacteria are spread on agar plates with increasing concentrations of antibiotics. Heteroresistance is determined as the growth of colonies at ≥8 × MIC of the main cell population. In the Etest assay, heteroresistance is determined by visible growth of colonies within the zone of clearing around the strips with a preformed continuous gradient of antibiotic concentrations. (b) Resistant bacteria that emerge before or during antibiotic treatment can grow in the presence of the antibiotic. They give rise to new populations distinct from the original ones. In the case of heteroresistance, resistant cells revert to the susceptible phenotype after the antibiotic treatment, usually due to high fitness costs. Persister cells can survive antibiotic treatment, but they cannot grow in the presence of the antibiotic. After the treatment, persisters can resume growth, switching back to the sensitive phenotype.
Figure 1. Heteroresistance detection methods and the difference between persister, resistant, and heteroresistant bacteria. (a) In the PAP assay, bacteria are spread on agar plates with increasing concentrations of antibiotics. Heteroresistance is determined as the growth of colonies at ≥8 × MIC of the main cell population. In the Etest assay, heteroresistance is determined by visible growth of colonies within the zone of clearing around the strips with a preformed continuous gradient of antibiotic concentrations. (b) Resistant bacteria that emerge before or during antibiotic treatment can grow in the presence of the antibiotic. They give rise to new populations distinct from the original ones. In the case of heteroresistance, resistant cells revert to the susceptible phenotype after the antibiotic treatment, usually due to high fitness costs. Persister cells can survive antibiotic treatment, but they cannot grow in the presence of the antibiotic. After the treatment, persisters can resume growth, switching back to the sensitive phenotype.
Ijms 23 00449 g001
Ijms 23 00449 g002 550
Figure 2. Two-component systems PhoPQ, PmrAB, and CrrAB control lipid A modifications by adding 4-amino-4-deoxy-l-arabinose (L-Ara4N) and phosphoethanolamine (PEtN). The modifications decrease the net negative charge of the lipid A, resulting in colistin resistance. See the text for more details.
Figure 2. Two-component systems PhoPQ, PmrAB, and CrrAB control lipid A modifications by adding 4-amino-4-deoxy-l-arabinose (L-Ara4N) and phosphoethanolamine (PEtN). The modifications decrease the net negative charge of the lipid A, resulting in colistin resistance. See the text for more details.
Ijms 23 00449 g002
Table
Table 1. Examples of heteroresistance in K. pneumoniae.
Table 1. Examples of heteroresistance in K. pneumoniae.
AntibioticSamplePrevalence of HeteroresistanceMechanisms of HeteroresistanceStability of Resistant PhenotypesReferences
ColistinMultidrug-resistant clinical isolates (worldwide)15/21NDND[48]
Carbapenemase-producing clinical strains (Greece)12/20NDStable (11)
Unstable (1)		[49]
Multidrug-resistant clinical isolate (South Africa)1/1Single amino acid change in PhoP, activation of the pmrHFIJKLM operonUnstable[50]
ESBL-producing clinical isolates5/13Mutations in the lpxM, mgrB, phoQ and yciM genesND[51]
Urine clinical isolate (Portugal)1/1Biofilm, small colony variant phenotypeND[52]
Stool clinical isolate (France)1/1Single nucleotide insertion in the mreB geneUnstable[53]
Clinical isolates (Hungary)68/140NDND[54]
Colistin susceptible K. pneu-moniae Ecl8 1/1Mutations in the crrB geneUnstable[55]
Multidrug-resistant urine isolates (USA)2/2Lower expression of mgrB, higher expression of phoPStable[44]
Clinical blood isolates (South Korea)3/252Different amino acid substitutions in PmrAB and PhoPQND[56]
ESBL-producing clinical isolates (Chile)8/60Diverse mutations in PmrAB and PhoPQ, disruption of the mgrB geneStable[57]
Clinical carbapenem-resistant isolates (South Korea)12/12Different amino acid substitutions in PmrAB and PhoPQStable[21]
CarbapenemsImipenemClinical isolates (Brazil, USA)8/15Carriage of the blaKPC gene, decreased expression of OmpK36Stable (6)
Unstable (2)		[58]
VIM-1-producing isolates (Spain)3/18NDND[59]
Clinical isolates (China)75/155NDND[60]
MeropenemClinical isolates (Greece)6/6Overexpression of the blaKPC-2 geneND[61]
OXA-48-producing isolates (Spain)24/24Decreased OmpK36 expression or activityStable[62]
Clinical isolates (China)38/155NDND[60]
TetracyclinesTigecyclineClinical isolates (China)21/334Overexpression of the acrAB genes, mutations in the ramR, soxR and rpsJ genesStable[63]
Carbapenem-resistant clinical isolates (China)49/95Upregulated expression of the pmrA, phoP and acrB genesND[64]
EravacyclineClinical isolates (China)20/393Mutation in the acrR or ramR genes, overexpression of the MacAB efflux pumpND[39]
AminoglycosidesAmikacinClinical isolates (China)13/155Increased expression of aac(6′)-Ib, aph (3′)-Ia, aac (3)-II
Mutations in parB, threonine dehydrogenase, Ssb, Sid		Unstable (11)
Stable (2)		[60]
AmikacinClinical isolates (Sweden)2/10Mutations: ubiJ Δ10 nt, PTS glucose transporter subunit IIA Δ1 nt, cydAΔ57 ntUnstable[22]
Gentamycin2/10aac(3)-IIa amplification
Tobramycin3/10aac(6′)-Ib-ct or aac(3)-IIa amplification
Netilmicin4/10aac(6′)-Ib-ct amplification
ChlorhexidineXDR clinical isolates (Israel)113/126NDND[65]
Prevalence of heteroresistance, number of heteroresistant isolates/total number of isolates; ND, not determined.
Table
Table 2. Drug combinations effective against heteroresistant K. pneumonia isolates.
Table 2. Drug combinations effective against heteroresistant K. pneumonia isolates.
IsolatesDrug CombinationReferences
MDR, colistin-heteroresistantColistin + meropenem[56]
Carbapenem-resistant, heteroresistant to polymyxin B, and resistant, heteroresistant or susceptible to tigecycline (4 isolates)Polymyxin B + tigecycline[64]
Carbapenem-resistant, heteroresistant to colistin and fosfomycinColistin + fosfomycin[85]
Carbapenem-resistant, heteroresistant to fosfomycin and ceftazidimeFosfomycin + ceftazidime
Pandrug-resistant, heteroresistant to fosfomycin and sulfamethoxazole/trimethoprimFosfomycin + sulfamethoxazole/trimethoprim
Pandrug-resistant, heteroresistant to amikacin and piperacillin/tazobactam Amikacin + piperacillin/tazobactam
Carbapenem-resistant, heteroresistant to polymyxin B (4 isolates)Polymyxin B + ceftazidime/avibactam [86]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Stojowska-Swędrzyńska, K.; Łupkowska, A.; Kuczyńska-Wiśnik, D.; Laskowska, E. Antibiotic Heteroresistance in Klebsiella pneumoniae. Int. J. Mol. Sci. 2022, 23, 449. https://doi.org/10.3390/ijms23010449

AMA Style

Stojowska-Swędrzyńska K, Łupkowska A, Kuczyńska-Wiśnik D, Laskowska E. Antibiotic Heteroresistance in Klebsiella pneumoniae. International Journal of Molecular Sciences. 2022; 23(1):449. https://doi.org/10.3390/ijms23010449

Chicago/Turabian Style

Stojowska-Swędrzyńska, Karolina, Adrianna Łupkowska, Dorota Kuczyńska-Wiśnik, and Ewa Laskowska. 2022. "Antibiotic Heteroresistance in Klebsiella pneumoniae" International Journal of Molecular Sciences 23, no. 1: 449. https://doi.org/10.3390/ijms23010449

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop