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Background As carbapenem-resistant Acinetobacter baumannii is dominant in clinical settings, the old polymyxin antibiotic colistin has been revived as a therapeutic option. The development of colistin resistance during treatment is becoming a growing concern. Objectives To access low- to mid-level colistin-resistant A. baumannii blood isolates recovered from an outbreak in a tertiary care hospital from a national antimicrobial surveillance study. Methods The entire bacterial genome was sequenced through long-read sequencing methodology. Quantitative RT–PCR was carried out to determine the level of gene expression. Relative growth rates were determined to estimate fitness costs of each isolate caused by the genetic alterations. Results The A. baumannii isolates belonged to global clone 2 harbouring two intrinsic phosphoethanolamine transferases. Cumulative alterations continuing the colistin resistance were observed. PmrC overproduction caused by the PmrBA226T alteration was identified in A. baumannii isolates with low-level colistin resistance and an additional PmrCR109H substitution led to mid-level colistin resistance. Truncation of the PmrC enzyme by insertion of ISAba59 was compensated by ISAba10-mediated overproduction of EptA and, in the last isolate, the complete PmrAB two-component regulatory system was eliminated to restore the biological cost of the bacterial host. Conclusions During the in-hospital outbreak, a trajectory of genetic modification in colistin-resistant A. baumannii isolates was observed for survival in the harsh conditions imposed by life-threatening drugs with the clear purpose of maintaining drug resistance above a certain level with a reasonable fitness cost.

Class D β-lactamases are composed of 14 families and the majority of the member enzymes are included in the OXA family. The genes for class D β-lactamases are frequently identified in the chromosome as an intrinsic resistance determinant in environmental bacteria and a few of these are found in mobile genetic elements carried by clinically significant pathogens. The most dominant OXA family among class D β-lactamases is superheterogeneous and the family needs to have an updated scheme for grouping OXA subfamilies through phylogenetic analysis. The OXA enzymes, even the members within a subfamily, have a diverse spectrum of resistance. Such varied activity could be derived from their active sites, which are distinct from those of the other serine β-lactamases. Their substrate profile is determined according to the size and position of the P-, Ω- and β5–β6 loops, assembling the active-site channel, which is very hydrophobic. Also, amino acid substitutions occurring in critical structures may alter the range of hydrolysed substrates and one subfamily could include members belonging to several functional groups. This review aims to describe the current class D β-lactamases including the functional groups, occurrence types (intrinsic or acquired) and substrate spectra and, focusing on the major OXA family, a new model for subfamily grouping will be presented.

Objectives A MALDI-TOF MS-based identification method for KPC-producing Enterobacterales was developed. Methods The molecular mass of the intact KPC-2 polypeptide was estimated for blaKPC-2 transformants using MALDI Microflex and the exact mass was confirmed by LC and a high-resolution MS/MS system. A total of 1181 clinical Enterobacterales strains, including 369 KPC producers and 812 KPC non-producers, were used to set up the methodology and the results were compared with those from PCR analyses. For external validation, a total of 458 Enterobacterales clinical isolates from a general hospital between December 2018 and April 2019 were used. Results The exact molecular mass of the intact KPC-2 protein was 28 718.13 Da and KPC peaks were observed at m/z 28 708.87–28 728.34 using MALDI Microflex. Most of the KPC-2 (99.1%, 335/338) and KPC-3 (100%, 6/6) producers presented a clear peak via this method, while 12.0% (3/25) of the KPC-4 producers had a peak of weak intensity associated with low levels of gene expression. It took less than 20 min for the entire assay to be performed with colonies on an agar plate. External validation showed that the analytical sensitivity and specificity of the method compared with PCR were 100% (59/59) and 99.50% (397/399), respectively. Conclusions The MALDI-TOF MS-based method for directly detecting the intact KPC protein is applicable to routine tests in clinical microbiology laboratories, supported by its speed, low cost and excellent sensitivity and specificity.

BackgroundTo assess the mortality dynamics of patients with Pseudomonas aeruginosa bloodstream infections (BSIs) and the influence of OprD deficiencies of the microorganism on early mortality.MethodsA prospective multicentre observational study was conducted with 120 patients with P. aeruginosa BSIs occurring between May 2016 and April 2017 in six general hospitals in South Korea. PCR and sequencing were carried out to identify the alterations in oprD and the presence of virulence factors. Cox regression was used to estimate the risk factors for mortality at each timepoint and Kaplan–Meier survival analyses were performed to determine the mortality dynamics.ResultsDuring the 6 week follow-up, 10.8% (13/120) of the patients with P. aeruginosa BSIs died in 2 weeks, 14.2% (17/120) in 4 weeks and 20.0% (24/120) in 6 weeks, revealing a steep decrease in cumulative survival between the fourth and sixth weeks. ICU admission and SOFA score were risk factors for mortality in any weeks after BSI onset and causative OprD-defective P. aeruginosa had a risk tendency for mortality within 6 weeks. Among the 120 P. aeruginosa blood isolates, 14 were XDR, nine produced either IMP-6 or VIM-2 MBL, and 21 had OprD deficiency.ConclusionsBSIs caused by OprD-defective P. aeruginosa resulted in a 2-fold higher 6 week mortality rate (33.3%) than that of BSIs caused by OprD-intact P. aeruginosa (17.2%), likely due to the decreased susceptibility to carbapenems and bacterial persistence in clinical settings.

OBJECTIVES The aac(6')-Ih gene encoding aminoglycoside 6'-N-acetyltransferase type I subtype h [AAC(6')-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination. METHODS Gene copy number was determined by quantitative PCR, expression by quantitative RT-PCR, MIC by microdilution and transfer by plasmid mobilization. RESULTS The aac(6')-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii PIS Aba23 promoters were provided by insertion of ISAba23, which disrupted the Pnative promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4. CONCLUSIONS We have found the origin of aac(6')-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.

Objectives To investigate the risk factors of patients with Klebsiella pneumoniae (KP) bloodstream infection (BSI) with a focus on antimicrobial resistance and virulence factors. Methods All KP BSI patients (n = 579) from six general hospitals during a 1 year period were included in this study. The risk factors of hosts and causative KP isolates were assessed to determine associations with the 30 day mortality of KP BSI patients by multivariate Cox hazards modelling. Results The 30 day mortality rate of KP BSI patients was 16.9% (98/579). Among the host-associated factors, increased SOFA score and leucopenia status exhibited strong associations with increased 30 day mortality. Among the pathogenic factors, carriage of the pks gene cluster (adjusted HR 1.80; 95% CI 1.16-2.79) was a risk factor, especially when accompanied by MDR. In this regard, KP isolates of the wzi50 capsular type (n = 22) frequently harboured pks (63.6%, 14/22) and ybtA (68.2%, n = 15) and mostly exhibited MDR (63.6%, n = 14), resulting in increased 30 day mortality. In contrast, hypermucoviscous KP isolates showed an inverse association with 30 day mortality (adjusted HR 0.55; 95% CI 0.33-0.90). Conclusions Despite the reported virulence of hypermucoviscous KP strains, they were associated with good prognoses in KP BSI patients. Importantly, carriage of the pks gene cluster, which is responsible for the synthesis of colibactin, was a relevant marker of early mortality.