Klebsiella pneumoniae is a rod-shaped, encapsulated, Gram-negative bacteria associated with multiple nosocomial infections. Multidrug-resistant (MDR) K. pneumoniae strains have been increasing and the therapeutic options are increasingly limited. Colistin is a long-used, polycationic, heptapeptide that has regained attention due to its activity against Gram-negative bacteria, including the MDR K. pneumoniae strains. However, this antibiotic has a complex mode of action that is still under research along with numerous side-effects. The acquisition of colistin resistance is mainly associated with alteration of lipid A net charge through the addition of cationic groups synthesized by the gene products of a multi-genic regulatory network. Besides mutations in these chromosomal genes, colistin resistance can also be achieved through the acquisition of plasmid-encoded genes. Nevertheless, the diversity of molecular markers for colistin resistance along with some adverse colistin properties compromises the reliability of colistin-resistance monitorization methods. The present review is focused on the colistin action and molecular resistance mechanisms, along with specific limitations on drug susceptibility testing for K. pneumoniae.

The effect of gamma, γ , radiation on the surface morphology and the optical properties of a prepared alpha-methyl curcumin: PMMA film were studied under different doses (7, 14, 21 and 28 Gy) of γ -rays emitted by ¹³⁷ Cs source. To monitor the effect of γ radiation on the prepared sample, the UV–visible absorbance and transmittance measurements of the pristine and the irradiated sample were carried out in the spectral range 400–900 nm at room temperature. An optical microscope and image J software were utilized to examine any changes in the surface morphology for the pre and post irradiated samples. The obtained results indicated that the surface morphology, linear and nonlinear optical properties of the films have been affected by γ radiation.

The evolutionary epidemiology, resistome, virulome and mobilome of thirty-one multidrug resistant Klebsiella pneumoniae clinical isolates from the northern Vila Real region of Portugal were characterized using whole-genome sequencing and bioinformatic analysis. The genomic population structure was dominated by two main sequence types (STs): ST147 (n = 17; 54.8%) and ST15 (n = 6; 19.4%) comprising four distinct genomic clusters. Two main carbapenemase coding genes were detected (bla_KPC-3 and bla_OXA-48) along with additional extended-spectrum β-lactamase coding loci (bla_CTX-M-15, bla_SHV-12, bla_SHV-27, and bla_SHV-187). Moreover, whole genome sequencing enabled the identification of one Klebsiella variicola KPC-3 producer isolate previously misidentified as K. pneumoniae, which in addition to the bla_KPC-3 carbapenemase gene, bore the chromosomal broad spectrum β-lactamase bla_LEN-2 coding gene, oqxAB and fosA resistance loci. The bla_KPC-3 genes were located in a Tn4401b transposon (K. variicolan = 1; K. pneumoniaen = 2) and Tn4401d isoform (K. pneumoniaen = 28). Overall, our work describes the first report of a bla_KPC-3 producing K. variicola, as well as the detection of this species during infection control measures in surveillance cultures from infected patients. It also highlights the importance of additional control measures to overcome the clonal dissemination of carbapenemase producing clones.

The emergence of carbapenemase-producing Klebsiella pneumoniae strains has triggered the use of old antibiotics such as colistin. This is driving the emergence of colistin resistance in multidrug-resistant strains that underlie life-threatening infections. This study analyses the mutational diversity of 22 genes associated with colistin resistance in 140 K. pneumoniae clinical isolates integrated in a high-resolution phylogenetic scenario. Colistin susceptibility was accessed by broth microdilution. A total of 98 isolates were susceptible and 16 were resistant, 10 of which were carbapenemase producers. Across the 22 genes examined, 171 non-synonymous mutations and 9 mutations associated with promoter regions were found. Eighty-five isolates had a truncation and/or deletion in at least one of the 22 genes. However, only seven mutations, the complete deletion of mgrB or insertion sequence (IS)-mediated disruption, were exclusively observed in resistant isolates. Four of these (mgrB^Ile13fs, pmrB^Gly207Asp, phoQ^His339Asp and ramA^Ile28Met) comprised novel mutations that are potentially involved in colistin resistance. One strain bore a ISEcp1-bla_CTX-M-15::mgrB disruption, underlying co-resistance to third-generation cephalosporins and colistin. Moreover, the high-resolution phylogenetic context shows that most of the mutational diversity spans multiple phylogenetic clades, and most of the mutations previously associated with colistin resistance are clade-associated and present in susceptible isolates, showing no correlation with colistin resistance. In conclusion, the present study provides relevant data on the genetic background of genes involved with colistin resistance deeply rooted across monophyletic groups and provides a better understanding of the genes and mutations involved in colistin resistance.

The emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the “skipped well” (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains (“very major errors”), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has high incidence in Portugal, causing severe and often fatal infections. Characterize the evolutionary history and epidemiology of CRKP in Portugal over a 40-year period. WGS was performed using the Illumina platform. In silico multilocus sequence typing, surface antigen characterization, and resistance gene detection were subsequently carried out. Core and pan-genome analyses were conducted using Roary. Genomic clusters (GCs) were identified based on a 21-SNP threshold. To estimate the divergence times of the most prevalent sequence types (ST) in the dataset, Bayesian evolutionary analysis was performed using BEAST. Nineteen GCs harboring carbapenemases were identified. The bla_KPC-3 gene was the most prevalent carbapenemase, linked to strains circulating in both hospital and community settings, with dissemination patterns at regional, interregional, and international levels. ST15 was the most established sequence type in Portugal, with nine distinct GCs identified in both clinical and environmental samples. Towards the end of 2010s, ST147 and ST13 were responsible for significant outbreaks associated with bla_KPC-3. This study underscores the value of genomic-based surveillance in understanding the evolution of high-risk clones coupled with the spread of AMR determinants. The data obtained highlights a shift in ST predominance across the country from an ST15-dominated period and strongly associated with ESBL dissemination, to the emergence of ST147 and ST13 CRKP clones, the latter associated with international transmission. This work further stresses the importance of cross-border surveillance efforts to monitor the emergence and dissemination of CRKP strains and inform risk assessment and prevention.