Three Klebsiella pneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all β-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K. pneumoniae isolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred to Escherichia coli strain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, IS Kpn23 , the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was named bla _BKC-1 . Brazilian Klebsiella carbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a β-lactamase of Sinorhizobium meliloti , an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very low k _cat values (0.0016 to 0.031 s ⁻¹ ). In fact, oxacillin was the best substrate for BKC-1 ( k _cat / K _m , 53,522.6 mM ⁻¹ s ⁻¹ ). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of β-lactamases in K. pneumoniae clinical isolates.

The emergence and rapid dissemination of colistin-resistant Escherichia coli carrying the plasmid-mediated mcr-1 gene have created an urgent need to develop specific screening methods. In this study, we evaluated four assays based on the inhibition of MCR-1 activity by EDTA: (i) a combined-disk test (CDT) comparing the inhibition zones of colistin and colistin (10 μg) plus EDTA (100 mM); (ii) reduction of colistin MIC (CMR) in the presence of EDTA (80 μg/ml); (iii) a modified rapid polymyxin Nordmann/Poirel test (MPNP); and (iv) alteration of zeta potential (R _ZP = ZP _+EDTA /ZP _−EDTA ). We obtained encouraging results for the detection of MCR-1 in E. coli isolates recovered from human, food, and animal samples, using the following assay parameters: ≥3 mm difference in the inhibition zones between colistin disks without and with EDTA; ≥4-fold colistin MIC decrease in the presence of EDTA; R _ZP of ≥2.5; and the absence of metabolic activity and proliferation, indicated by unchanged color of phenol red in the presence of colistin-EDTA, in the MPNP test. In this regard, the CDT, CMR, R _ZP , and MPNP assays exhibited sensitivities of 96.7, 96.7, 95.1, and 96.7% and specificities of 89.6, 83.3, 100, and 100%, respectively, for detecting MCR-1-positive E. coli . Our results demonstrate that inhibition by EDTA and zeta potential assays may provide simple and inexpensive methods for the presumptive detection of MCR-1-producing E. coli isolates in human and veterinary diagnostic laboratories.

Bacteriophages are the most abundant organisms on Earth. As there are few effective treatment options against some pathogens, the interest in the bacteriophage control of multi-drug-resistant bacterial pathogens is escalating, especially for Klebsiella pneumoniae. This study aimed to develop a phage-based solution to the rising incidence of extensively drug-resistant clinical Klebsiella pneumoniae sequence type (ST16) infections starting from a set of phages recently characterized against this lineage. A phage-cocktail (Katrice-16) composed of eight lytic phages was characterized for potential use in humans. In vitro and in vivo broth inhibition and Galleria mellonella rescue assays were used to demonstrate the efficacy of this approach using a collection of 56 strains of K. pneumoniae ST16, with distinct genetic backgrounds that were collected from clinical infections from four disparate nations. Additionally, Katrice-16 anti-biofilm activity, synergism with meropenem, and activity in human body fluids were also assessed. Katrice-16 was highly active in vitro against our K. pneumoniae ST16 collection (AUC% median = 86.48%; Q1 = 83.8%; Q2 = 96.85%; Q3 = 98.85%). It additionally demonstrated excellent in vivo activity in G. mellonella rescue assays, even with larvae infected by isolates that exhibited moderate in vitro inhibition. We measured significant anti-biofilm activity over 12 h (p = .0113) and synergic activity with meropenem. In addition, we also demonstrate that Katrice-16 maintained high activity in human body fluids. Our results indicate that our cocktail will likely be an effective solution for human infections with this increasingly prevalent and often highly resistant bacterial clone.

The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13pH), magnesium deprivation (Kp13Mg), high concentrations of calcium (Kp13Ca) and iron (Kp13Fe), and a control condition with PB (Kp13PolB). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.