In Vitro Activity of “Old” and “New” Antimicrobials against the Klebsiella pneumoniae Complex

The Klebsiella pneumoniae complex is a commonly isolated bacteria in human infections. These opportunistic pathogens pose a serious threat to public health due to their potential transmission to the human population. Resistance to carbapenems is a significant antimicrobial resistance mechanism, leading to limited therapeutic options. Therefore, the aim of this study was to evaluate the in vitro activity of fosfomycin, colistin, ceftazidime–avibactam, and meropenem–vaborbactam against multidrug-resistant K. pneumoniae complex strains. This study involved 160 strains of Gram-negative rods, comprising 138 K. pneumoniae and 22 K. variicola. The minimal inhibitory concentration of fosfomycin was estimated using the agar dilution method, and for colistin, the microdilution method was employed. Susceptibility to ceftazidime–avibactam and meropenem–vaborbactam was determined using the gradient strip method. All analyzed K. pneumoniae complex isolates produced extended-spectrum β-lactamases, and 60.0% exhibited carbapenemases. The majority of the analyzed strains were susceptible to fosfomycin and colistin (62.5%). Among pandrug-resistant K. pneumoniae complex isolates, the highest susceptibility was observed with colistin (43.9%). Fosfomycin demonstrated good activity against ESβLs- and VIM-positive isolates from this complex. Colistin also exhibited satisfactory in vitro activity against VIM- and KPC-positive isolates from the K. pneumoniae complex. Ceftazidime–avibactam displayed good activity against K. pneumoniae complex strains producing ESβLs, KPC, and OXA enzymes. Additionally, meropenem–vaborbactam showed satisfactory in vitro activity against ESβLs- and KPC-positive isolates from this complex.


Introduction
The Klebsiella pneumoniae complex is a common causative bacteria for both communityand hospital-acquired infections.The predominant and frequently isolated species within this complex is K. pneumoniae.The incidence of these bacteria causing severe infections with substantial morbidity and mortality has been steadily rising [1].Notably, in recent years, Klebsiella variicola has been increasingly identified, particularly in cases of bloodstream infections [2,3].Both K. pneumoniae and K. variicola are associated with severe infections leading to high mortality rates, especially in immunocompromised patients [1,4].Furthermore, these bacteria frequently exhibit diverse mechanisms of antimicrobial resistance.For many years, the predominant and critical mechanism of antimicrobial resistance within the K. pneumoniae complex was the production of extended-spectrum β-lactamases (ESβLs).However, in the past decade, more and more reports have appeared describing the identification of strains producing carbapenemases [5].Strains producing EsβLs and carbapenemases can colonize the gastrointestinal tract of healthy individuals, subsequently leading to serious infections.Within the K. pneumoniae isolates, three classes of carbapenemases are identified: class A (Klebsiella pneumoniae carbapenemases, KPC), class B (metallo-β-lactamases, MβL), and class D (oxacillinases, OXA) [6].Strains producing KPC enzymes are frequently isolated in the United States and Israel [7].In Poland, the first KPC-positive K. pneumoniae strain was isolated in 2008.Between 2019 and 2021, the frequency of isolation increased by around 200% and accounted for 18% of all cases.[8,9].The first MβL-positive K. pneumoniae strain in Poland was isolated in 2006 and identified as the VIM-1 enzyme [10].Since the isolation of the first strain in Poland, VIM enzymes have been identified in various species, with the two dominant ones being Enterobacter hormaechei and Klebsiella oxytoca.However, in recent years, there has been a shift, and an increase in the frequency of isolation of K. pneumoniae and Escherichia coli strains has been observed [9].Notably, VIM-positive strains were the only carbapenemase-producing isolates for which no increase was recorded in Poland in 2020-2021.The first NDM-positive strain appeared in Poland in 2012 and rapidly spread to other hospitals, causing local epidemics.In 2021, NDM-positive strains accounted for 73% of all carbapenemase-producing Enterobacterales strains in Poland, reflecting a substantial increase in incidence (of 99%) compared to 2019 [9].In Poland, the first OXA-48-positive isolate, identified as E. hormaechei subsp.steigerwaltii, was recognized in 2012.Until 2017, OXA-48-positive strains were relatively infrequently isolated in Poland.However, in 2021, there was an observed increase in the frequency of strains with this phenotype by approximately 70% compared to 2019.The OXA-48-like group of carbapenemases includes various enzymes such as OXA-48, OXA-181, OXA-232, OXA-204, OXA-162, and OXA-244 [11].Although carbapenemases can be produced by all Enterobacterales, K. pneumoniae remains the primary species in which they are identified.According to data from the European Antimicrobial Resistance Surveillance System (EARSS), in Poland, the proportion of K. pneumoniae strains resistant to carbapenems isolated from invasive infections increased from 0.8% in 2012 to 6.4% in 2017 and further rose to 16.8% in 2022 [5].The prevalence of carbapenem-resistant K. pneumoniae strains in Europe varies, ranging from 0.0% in Finland and Iceland to 72.0% in Greece, according to EARSS data from 2022 [5].Additionally, strains producing more than one β-lactamase from different groups are increasingly being identified.These include combinations such as ESβLs and carbapenemases or two different carbapenemases.In such scenarios, therapeutic options become highly limited.Information regarding the antimicrobial resistance profile of K. variicola strains is scant, often limited to case reports.
Fosfomycin, colistin, ceftazidime-avibactam, and meropenem-vaborbactam are registered for treating severe infections caused by strains with limited therapeutic alternatives.The inclusion of these antimicrobials is justified for various reasons.Fosfomycin and colistin possess unique and distinct mechanisms of action, exhibiting bactericidal activity against a broad spectrum of bacterial species.Additionally, fosfomycin demonstrates excellent diffusion in body tissues and has low toxicity.While monotherapy with fosfomycin is not recommended for treating infections caused by MβL-producing Enterobacterales, it exhibits synergy with other antimicrobials, such as ceftazidime-avibactam [12,13].On the other hand, despite its high toxicity, colistin remains a drug of last resort for treating multidrugresistant (MDR) bacterial infections, including those caused by K. pneumoniae [14,15].The introduction of new combinations of β-lactams with β-lactamase inhibitors has significantly broadened therapeutic options for treating infections caused by carbapenemase-producing strains.These drugs are deemed safe for patients and exhibit effective tissue penetration.Avibactam, a diazabicyclooctane non-β-lactam, binds covalently and reversibly to β-lactamases.Its crucial advantage lies in its ability to inhibit ESβLs, AmpC β-lactamases, as well as class A and D carbapenemases [16].Vaborbactam, the first boronic acid βlactamase inhibitor, belongs to a group known for the reversible and competitive inhibition of serine β-lactamases.It effectively inhibits class A β-lactamases, including KPC carbapenemases, and class C β-lactamases.However, it does not inhibit class B and class D β-lactamases [16].
The objective of this study was to compare the in vitro activity of fosfomycin, colistin, ceftazidime-avibactam, and meropenem-vaborbactam against MDR K. pneumoniae complex, encompassing isolates producing ESβLs and various carbapenemases.

MIC Determination
Among the 160 K. pneumoniae complex strains, 100 (62.5%) were susceptible to fosfomycin and colistin, 99 (58.1%) to ceftazidime-avibactam, and 88 (55.0%) to meropenemvaborbactam.The detailed susceptibility profiles for the specific groups of the analyzed K. pneumoniae complex strains are presented in Figure 1.

MIC Determination
Among the 160 K. pneumoniae complex strains, 100 (62.5%) were susceptible to fosfomycin and colistin, 99 (58.1%) to ceftazidime-avibactam, and 88 (55.0%) to meropenem-vaborbactam.The detailed susceptibility profiles for the specific groups of the analyzed K. pneumoniae complex strains are presented in Figure 1.
The K. variicola ESβLs-positive strains exhibited the lowest MIC 50 and MIC 90 values for all tested antimicrobials, with the exception of the MIC 50 for colistin, which had the highest value among all analyzed groups.Detailed data on MIC 50 and MIC 90 values are provided in Table 2. Fosfomycin, colistin, ceftazidime-avibactam, and meropenem-vaborbactam MICs distributions for the specific groups of strains are presented in Tables 3-5.The MICs of fosfomycin ranged from 4 to >256 mg/L for PDR, carbapenemase-positive, and ESβLs-positive K. pneumoniae isolates, and from 4 to 128 mg/L for K. variicola ESβLspositive isolates.
The MICs of colistin ranged from 0.25 to >16 mg/L for PDR and carbapenemasepositive K. pneumoniae isolates, from 0.25 to 16 mg/L for ESβLs-positive K. pneumoniae isolates, and from 0.25 to 8 mg/L for K. variicola ESβLs-positive isolates.
The MICs of ceftazidime-avibactam ranged from 0.25 to 256 mg/L for PDR isolates, from 0.38 to 256 mg/L for carbapenemase-positive K. pneumoniae isolates, from 0.047 to 64 mg/L for ESβLs-positive K. pneumoniae isolates, and from 0.047 to 2 mg/L for K. variicola ESβLs-positive isolates.The MICs of meropenem-vaborbactam ranged from 0.064 to 256 mg/L for PDR strains, from 0.032 to >256 mg/L for carbapenemase-positive strains, from 0.032 to >256 mg/L for ESβLs-positive K. pneumoniae strains, and from 0.032 to 2 mg/L for K. variicola ESβLs-positive strains.

Bacterial Strains
The study included 160 non-replicated strains of the K. pneumoniae complex isolated from various clinical samples derived from patients at Dr. A. Jurasz University Hospital No. 1 in Bydgoszcz, Poland.These isolates were collected over a one-year period, spanning from 1 October 2022 to 30 September 2023.The strains were obtained through standard diagnostic procedures at the Microbiology Department of Dr. A. Jurasz University Hospital No. 1 in Bydgoszcz, Poland.

Identification and Susceptibility Testing
Isolate identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the MALDI TOF Biotyper Microflex LT/SH system (Bruker, Bremen, Germany) with version 7.0.0.1 software.For strains with a value ≥2.300, identification was conducted once, while for isolates with values between 2.000 and 2.299, identification was performed thrice.
Antimicrobial susceptibility tests for all the analyzed strains were conducted using the Phoenix M50 system (Becton Dickinson, NJ, USA) with NMIC-408 panels.The obtained results were automatically interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 Recommendations (v 13.0) [17].An isolate was classified as MDR if it demonstrated non-susceptibility to ≥1 agent in >3 antimicrobial categories and as pandrug-resistant (PDR) if it exhibited non-susceptibility to all antimicrobial agents [18].The definitions of MDR and PDR were determined based on the results obtained from Phoenix.

Discussion
The escalating issue of bacterial resistance to antimicrobials has prompted the exploration of new drugs and the reconsideration of well-established ones.Fosfomycin and colistin, discovered a few decades ago, have regained attention.The introduction of intravenous fosfomycin in 2019, particularly in European countries like Poland, broadened its utility against infections caused by MDR strains.Colistin, despite its historical use, faced limitations due to toxicity after 1970.However, both drugs offer a broad spectrum of activity, encompassing strains producing β-lactamases from classes A to D. While various studies have addressed the activity of fosfomycin and colistin, a notable gap exists concerning isolates from Eastern Europe, including Poland.The results of this study demonstrated that over 62% of the analyzed K. pneumoniae complex strains in Poland were susceptible to fosfomycin.Notably, a prior study by Kowalska-Krochmal et al. [20] on a substantial cohort of Klebsiella spp.isolates (250 strains) from patients with invasive infections in Poland reported a 66.0% susceptibility to fosfomycin, with MIC 50 at 32 mg/L and MIC 90 at 512 mg/L.In the analyzed group, 86 isolates were ESβLs-positive, 26 were ESβLs-and carbapenemase-positive, and 58 were carbapenemase-positive.The results of this study indicated that fosfomycin exhibited high potency, with over 86% susceptibility, against K. pneumoniae VIM-positive isolates and K. variicola ESβLs-positive isolates.These findings underscore the importance of considering regional variations in antimicrobial susceptibility, and obtained results contribute valuable insights into the efficiency of fosfomycin against specific strains in Poland.
According to research conducted in Turkey [21], 53.2% of Klebsiella spp.isolates were susceptible to fosfomycin.Notably, the highest susceptibility was observed for OXA-48and NDM-positive strains, with 73.3% being susceptible, while strains producing only NDM enzymes exhibited the lowest susceptibility at 33.3%.In this study, none of the nine analyzed OXA-positive isolates were susceptible to fosfomycin, with only two isolates producing OXA-48 and seven strains producing OXA-181.In contrast, a study by Demirci-Duarte et al. [21] reported that 50.5% of OXA-48-positive Klebsiella spp.isolates (n = 104) were susceptible to fosfomycin.Additionally, Aprile et al. [22] found that 76% of KPCpositive K. pneumoniae strains were susceptible to fosfomycin, while none of the NDM-and OXA-48-positive strains showed susceptibility.However, in this study, the value for KPCpositive strains was slightly lower, around 64%, although the number of strains analyzed was smaller.A further study from Latin America [23] reported varying susceptibility of K. pneumoniae strains to fosfomycin, ranging from 90.9% for strains isolated in Chile to 100% for strains isolated in Mexico, with a total of 601 K. pneumoniae isolates included.For carbapenem-non-susceptible K. pneumoniae strains (183), the susceptibility values were 71.4% and 100%, respectively.On the other hand, Zarakolu et al. [24] reported that 90.7% of carbapenem-susceptible K. pneumoniae isolates and 69.4% of carbapenem-resistant isolates were susceptible to fosfomycin.
The results of this study revealed that over 62% of the analyzed K. pneumoniae complex strains were susceptible to colistin.Comparable findings were reported in Greece [25], where the antimicrobial activity of colistin demonstrated a susceptibility rate of 64% among 392 carbapenem-resistant K. pneumoniae strains.In Poland, Pruss et al. [26] conducted a study on a group of 200 K. pneumoniae isolated from clinical samples.Among the analyzed ESβLs-positive strains, they were all susceptible to colistin.For carbapenemase-positive isolates, the susceptibility varied based on the phenotype.The highest susceptibility to colistin was obtained for KPC-positive isolates (100%), followed by NDM-positive isolates (82.3%), and only 50.0% for OXA-48-positive strains.The results of this study showed that colistin was highly potent (≥80% susceptibility) against KPC-positive and VIM-positive K. pneumoniae isolates.However, the lowest susceptibility to colistin was noted for OXApositive strains, with only 33.4% susceptibility, including the OXA-48 and OXA-181 strains.Research conducted in European countries, including Poland [27], reported that 95.6% of K. pneumoniae among 4201 isolates were susceptible to colistin, with MIC 50/90 values of 1 mg/L, ranging from ≤0.12 mg/L to >4 mg/L.Another study from Spain [28] found that the susceptibility of carbapenemase-producing K. pneumoniae strains to colistin decreased from 86.5% to 68.3% over three years.Interestingly, a substantial difference in colistin MIC 90 values was observed among carbapenemase-producing K. pneumoniae isolates in Greece, India, and Poland, with values of 64 mg/L, 32 mg/L, and 8 mg/L, respectively.Meanwhile, colistin MIC 50 values were similar, at 1 mg/L, 0.5 mg/L, and 0.75 mg/L, respectively.
There are no data in the literature regarding the frequency of the occurrence of colistinsusceptible K. variicola isolates.However, a few articles describe K. variicola strains resistant to this antimicrobial [29,30].In a previous study, among 13 K. variicola strains isolated from clinical samples, 46.1% exhibited resistance to colistin (unpublished data).
Ceftazidime-avibactam and meropenem-vaborbactam are combinations of established β-lactams with new β-lactamase inhibitors.These antimicrobials received approval from the US Food and Drug Administration and the European Medicines Agency several years ago.Both ceftazidime-avibactam and meropenem-vaborbactam exhibit activity against strains producing class A and C β-lactamases.Additionally, ceftazidime-avibactam demonstrates efficacy against strains producing class D β-lactamases.
The results of this study demonstrate that ceftazidime-avibactam and meropenemvaborbactam were highly effective (>88% susceptibility) against K. pneumoniae KPC-positive isolates, as well as K. variicola and K. pneumoniae ESβLs-positive isolates.Previous studies from this medical center [31] have also confirmed the high in vitro activity of ceftazidimeavibactam against K. pneumoniae strains producing ESβLs enzymes.In the cited study, out of four strains producing carbapenemases (two OXA-48 and two KPC), three were susceptible to ceftazidime-avibactam.A study from Latin America [23] found that the susceptibility of K. pneumoniae strains to ceftazidime-avibactam varied from 87.0% for strains isolated in Brazil to 100% for strains isolated in Mexico.For carbapenem-nonsusceptible K. pneumoniae strains (n = 183), the values were 83.3% and 100%, respectively, with the lowest value obtained in Colombia at 68.6%.According to research conducted in European countries, including Poland [27], 98.9% of K. pneumoniae of 4201 isolates were susceptible to ceftazidime-avibactam with MIC 50 0.12 mg/L and MIC 90 1 mg/L, ranging from ≤0.015 mg/L to >128 mg/L.Another study from Pittsburgh [32] noted that the susceptibility of K. pneumoniae strains to ceftazidime-avibactam achieved 79.0%.All of the analyzed isolates were carbapenem-resistant, and 93% of them produced KPC enzymes.In this study, over 35% of carbapenemase-positive K. pneumoniae strains were susceptible to ceftazidime-avibactam.Among KPC-positive strains, all were susceptible to this drug.Additionally, a high percentage of OXA-positive K. pneumoniae strains were susceptible to ceftazidime-avibactam, almost 89%.In contrast, Bianco et al. [33] analyzed susceptibility to selected antimicrobials in seven multi-carbapenemase K. pneumoniae isolates, and none of them were susceptible to ceftazidime-avibactam.Similar results were obtained in this study, including 15 multi-carbapenemase K. pneumoniae strains.
According to research conducted in US medical centers between 2018 and 2022 [34], 97.1% of 7153 K. pneumoniae isolates were susceptible to meropenem-vaborbactam.Another study from Pittsburgh [32] noted that the susceptibility of carbapenem-resistant K. pneumoniae strains to meropenem-vaborbactam reached 99.0%, with MICs ranging from ≤0.015 mg/L to >32 mg/L.The majority of these strains (39%) were KPC-positive.Gaibani et al. [35] noted that 87% of KPC-positive K. pneumoniae strains isolated from bloodstream infections in a hospital in Bologna were susceptible to meropenem-vaborbactam.In this study, over 30% of carbapenemase-positive K. pneumoniae strains were susceptible to meropenem-vaborbactam.Among the KPC-positive strains, all were susceptible to this drug.In contrast, Bianco et al. [33] analyzed susceptibility to selected antimicrobials in multi-carbapenemase K. pneumoniae isolates.Two out of seven strains were susceptible to meropenem-vaborbactam.However, in this study, which included 15 multicarbapenemase isolates, none of the analyzed K. pneumoniae strains were susceptible to meropenem-vaborbactam.
None of the analyzed K. variicola strains produced carbapenemases, but there are articles in the literature about carbapenemase-positive K. variicola strains that produce NDM, KPC, IMP, or OXA enzymes [36][37][38][39].Although there are a few reports in the literature assessing the susceptibility of K. variicola isolates to antimicrobials, they did not include fosfomycin or new combinations of β-lactams with β-lactamase inhibitors.The limited number of articles on the susceptibility of K. variicola to antimicrobials may be related to the challenges in identifying this species using automatic methods.Additionally, in the mass spectrometry method, the identification result depends on the number and type of spectra collected in the virtual library, making misidentification to other species within the complex possible.
In the era of increasing bacterial resistance to antimicrobials, rapid identification of carbapenemase-positive strains is a microbiological, clinical, and epidemiological concern.The emergence of MDR isolates often prompts difficult choices, such as using drugs with reduced susceptibility or increasing the drug dose.Therefore, it is crucial to monitor the emergence of resistant strains and quickly detect resistance to both "old" and "new" antimicrobials.
However, this study has some limitations.First, all strains were isolated from patients with symptoms of infection, and antimicrobial susceptibility testing was performed in vitro, making it unknown whether the same results would be obtained in vivo.Second, the strains came from patients in one hospital.Third, all K. variicola isolates in the study exhibited only an ESβL phenotype.Considering these aspects, further research should be continued and its scope expanded.

Conclusions
MDR K. pneumoniae complex infections, particularly those involving carbapenemaseproducing strains, are becoming increasingly common.Fosfomycin, colistin, ceftazidimeavibactam, and meropenem-vaborbactam appear to be promising antimicrobials for treating infections caused by the MDR and PDR K. pneumoniae complex.The results obtained in this study underscore the importance of identifying carabapenemase.The most prevalent mechanisms of carbapenem resistance were NDM and OXA-181 enzymes.
Based on the obtained results, the in vitro activity of fosfomycin is quite satisfactory against VIM-positive K. pneumoniae isolates and ESβLs-positive K. variicola isolates.Colistin demonstrated the highest in vitro antimicrobial activity against strains producing KPC and VIM carbapenemases.However, new combinations of β-lactams with βlactamase inhibitors (ceftazidime-avibactam and meropenem-vaborbactam) presented excellent in vitro activity against EsβLs-and KPC-positive isolates.Currently, strains producing more than one carbapenemase pose a significant challenge.The findings of this study indicate that only colistin showed good, but unsatisfactory, in vitro activity against K. pneumoniae complex strains.Therefore, the susceptibility of strains to any antimicrobial agents is not constant over time and should be monitored continuously.