Whole Genome Multi-Locus Sequence Typing and Genomic Single Nucleotide Polymorphism Analysis for Epidemiological Typing of Pseudomonas aeruginosa From Indonesian Intensive Care Units.

We have previously studied carbapenem non-susceptible Pseudomonas aeruginosa (CNPA) strains from intensive care units (ICUs) in a referral hospital in Jakarta, Indonesia (Pelegrin et al., 2019). We documented that CNPA transmissions and acquisitions among patients were variable over time and that these were not significantly reduced by a set of infection control measures. Three high risk international CNPA clones (sequence type (ST)235, ST823, ST357) dominated, and carbapenem resistance was due to carbapenemase-encoding genes and mutations in the porin OprD. Pelegrin et al. (2019) reported core genome analysis of these strains. We present a more refined and detailed whole genome-based analysis of major clones represented in the same dataset. As per our knowledge, this is the first study reporting Single Nucleotide Polymorphisms (wgSNP) analysis of Pseudomonas strains. With whole genome-based Multi Locus Sequence Typing (wgMLST) of the 3 CNPA clones (ST235, ST357 and ST823), three to eleven subgroups with up to 200 allelic variants were observed for each of the CNPA clones. Furthermore, we analyzed these CNPA clone clusters for the presence of wgSNP to redefine CNPA transmission events during hospitalization. A maximum number 35350 SNPs (including non-informative wgSNPs) and 398 SNPs (ST-specific_informative-wgSNPs) were found in ST235, 34,570 SNPs (including non-informative wgSNPs) and 111 SNPs (ST-specific_informative-wgSNPs) in ST357 and 26,443 SNPs (including non-informative SNPs) and 61 SNPs (ST-specific_informative-wgSNPs) in ST823. ST-specific_Informative-wgSNPs were commonly noticed in sensor-response regulator genes. However, the majority of non-informative wgSNPs was found in conserved hypothetical proteins or in uncharacterized proteins. Of note, antibiotic resistance and virulence genes segregated according to the wgSNP analyses. A total of 8 transmission chains for ST235 strains followed by 9 and 4 possible transmission chains for ST357 and ST823 were traceable on the basis of pairwise distances of informative-wgSNPs (0 to 4 SNPs) among the strains. The present study demonstrates the value of detailed whole genome sequence analysis for highly refined epidemiological analysis of P. aeruginosa.

Pseudomonas aeruginosa: a clinical and genomics update.

Antimicrobial resistance (AMR) has become a global medical priority that needs urgent resolution. Pseudomonas aeruginosa is a versatile, adaptable bacterial species with widespread environmental occurrence, strong medical relevance, a diverse set of virulence genes and a multitude of intrinsic and possibly acquired antibiotic resistance traits. Pseudomonas aeruginosa causes a wide variety of infections and has an epidemic-clonal population structure. Several of its dominant global clones have collected a wide variety of resistance genes rendering them multi-drug resistant (MDR) and particularly threatening groups of vulnerable individuals including surgical patients, immunocompromised patients, Caucasians suffering from cystic fibrosis (CF) and more. AMR and MDR especially are particularly problematic in P. aeruginosa significantly complicating successful antibiotic treatment. In addition, antimicrobial susceptibility testing (AST) of P. aeruginosa can be cumbersome due to its slow growth or the massive production of exopolysaccharides and other extracellular compounds. For that reason, phenotypic AST is progressively challenged by genotypic methods using whole genome sequences (WGS) and large-scale phenotype databases as a framework of reference. We here summarize the state of affairs and the quality level of WGS-based AST for P. aeruginosa mostly from clinical origin.

Abundance of Colistin-Resistant, OXA-23- and ArmA-Producing Acinetobacter baumannii Belonging to International Clone 2 in Greece.

Carbapenem resistant Acinetobacter baumannii (CRAB) represents one of the most challenging pathogens in clinical settings. Colistin is routinely used for treatment of infections by this pathogen, but increasing colistin resistance has been reported. We obtained 122 CRAB isolates from nine Greek hospitals between 2015 and 2017, and those colistin resistant (ColR; N = 40, 32.8%) were whole genome sequenced, also by including two colistin susceptible (ColS) isolates for comparison. All ColR isolates were characterized by a previously described mutation, PmrBA226V, which was associated with low-level colistin resistance. Some isolates were characterized by additional mutations in PmrB (E140V or L178F) or PmrA (K172I or D10N), first described here, and higher colistin minimum inhibitory concentrations (MICs), up to 64 mg/L. Mass spectrometry analysis of lipid A showed the presence of a phosphoethanolamine (pEtN) moiety on lipid A, likely resulting from the PmrA/B-induced pmrC overexpression. Interestingly, also the two ColS isolates had the same lipid A modification, suggesting that not all lipid A modifications lead to colistin resistance or that other factors could contribute to the resistance phenotype. Most of the isolates (N = 37, 92.5%) belonged to the globally distributed international clone (IC) 2 and comprised four different sequence types (STs) as defined by using the Oxford scheme (ST 425, 208, 451, and 436). Three isolates belonged to IC1 and ST1567. All the genomes harbored an intrinsic bla OXA-51 group carbapenemase gene, where bla OXA-66 and bla OXA-69 were associated with IC2 and IC1, respectively. Carbapenem resistance was due to the most commonly reported acquired carbapenemase gene bla OXA-23, with ISAba1 located upstream of the gene and likely increasing its expression. The armA gene, associated with high-level resistance to aminoglycosides, was detected in 87.5% of isolates. Collectively, these results revealed a convergent evolution of different clonal lineages toward the same colistin resistance mechanism, thus limiting the effective therapeutic options for the treatment of CRAB infections.

Genomic Epidemiology of Carbapenem- and Colistin-Resistant Klebsiella pneumoniae Isolates From Serbia: Predominance of ST101 Strains Carrying a Novel OXA-48 Plasmid.

Klebsiella pneumoniae is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical K. pneumoniae isolates from Serbia. Consecutive non-replicate K. pneumoniae clinical isolates (n = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem (n = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates (N = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, N = 38), CG258 (ST437, N = 4; ST340, N = 1; ST258, N = 1) and CG17 (ST336, N = 1). mcr genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrBC28S being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the bla OXA- 48 genetic background. The bla OXA- 48 gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum ß-lactamase-encoding gene bla CTX-M-15 and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2∗, K3∗, Q30∗, adenine deletion leading to frameshift and premature termination, IS5-mediated inactivation) and expressed different carbapenemases: OXA-48 (ST437 and ST336), NDM-1 (ST437 and ST340) and KPC-2 (ST258). Our study reports the clonal expansion of the newly emerging ST101 clone in Serbia. This high-risk clone appears adept at acquiring resistance, and efforts should be made to contain the spread of such clone.

Differences and overlaps between Phd studies in diagnostic microbiology in industrial and academic settings.

Industrial and academic needs for innovation and fundamental research are essential and not widely different. Depending on the industrial setting, research and development (R&D) activities may be more focused on the developmental aspects given the need to ultimately sell useful products. However, one of the biggest differences between academic and industrial R&D will usually be the funding model applied and the priority setting between innovative research and product development. Generalizing, companies usually opt for development using customer- and consumer-derived funds whereas university research is driven by open innovation, mostly funded by taxpayer's money. Obviously, both approaches require scientific rigor and quality, dedication and perseverance and obtaining a PhD degree can be achieved in both settings. The formal differences between the two settings need to be realized and students should make an educated choice prior to the start of PhD-level research activities. Intrinsic differences in scientific approaches between the two categories of employers are not often discussed in great detail. We will here document our experience in this field and provide insights into the need for purely fundamental research, industrial R&D and current mixed models at the level of European funding of research. The field of diagnostics in clinical bacteriology and infectious diseases will serve as a source of reference.

High-Risk International Clones of Carbapenem-Nonsusceptible Pseudomonas aeruginosa Endemic to Indonesian Intensive Care Units: Impact of a Multifaceted Infection Control Intervention Analyzed at the Genomic Level.

Infection control effectiveness evaluations require detailed epidemiological and microbiological data. We analyzed the genomic profiles of carbapenem-nonsusceptible Pseudomonas aeruginosa (CNPA) strains collected from two intensive care units (ICUs) in the national referral hospital in Jakarta, Indonesia, where a multifaceted infection control intervention was applied. We used clinical data combined with whole-genome sequencing (WGS) of systematically collected CNPA to infer the transmission dynamics of CNPA strains and to characterize their resistome. We found that the number of CNPA transmissions and acquisitions by patients was highly variable over time but that, overall, the rates were not significantly reduced by the intervention. Environmental sources were involved in these transmissions and acquisitions. Four high-risk international CNPA clones (ST235, ST823, ST357, and ST446) dominated, but the distribution of these clones changed significantly after the intervention was implemented. Using resistome analysis, carbapenem resistance was explained by the presence of various carbapenemase-encoding genes (blaGES-5, blaVIM-2-8, and blaIMP-1-7-43) and by mutations within the porin OprD. Our results reveal for the first time the dynamics of P. aeruginosa antimicrobial resistance (AMR) profiles in Indonesia and additionally show the utility of WGS in combination with clinical data to evaluate the impact of an infection control intervention. (This study has been registered at www.trialregister.nl under registration no. NTR5541).IMPORTANCE In low-to-middle-income countries such as Indonesia, work in intensive care units (ICUs) can be hampered by lack of resources. Conducting large epidemiological studies in such settings using genomic tools is rather challenging. Still, we were able to systematically study the transmissions of carbapenem-nonsusceptible strains of P. aeruginosa (CNPA) within and between ICUs, before and after an infection control intervention. Our data show the importance of the broad dissemination of the internationally recognized CNPA clones, the relevance of environmental reservoirs, and the mixed effects of the implemented intervention; it led to a profound change in the clonal make-up of CNPA, but it did not reduce the patients' risk of CNPA acquisitions. Thus, CNPA epidemiology in Indonesian ICUs is part of a global expansion of multiple CNPA clones that remains difficult to control by infection prevention measures.

Epidemiology and characterisation of carbapenem-non-susceptible Pseudomonas aeruginosa in a large intensive care unit in Jakarta, Indonesia.

The aim of this study was to describe the epidemiology and clinical impact of carbapenem-non-susceptible Pseudomonas aeruginosa (CNPA) in intensive care units (ICUs) of the national referral hospital of Indonesia. Adult patients admitted to ICUs were prospectively included. Pseudomonas aeruginosa were from clinical cultures and systematic screening. Environmental niches and healthcare workers (HCWs) were also screened. Susceptibility was determined phenotypically and the presence of carbapenemase genes was determined by PCR. Multiple loci variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) were used for genotyping. Of the patients included in the study, 17/412 (4.1%) carried CNPA on admission and 34/395 (8.6%) became positive during their ICU stay. The acquisition rate was 18/1000 patient-days at risk. Of 16 environmental isolates, 12 (75.0%) were CNPA. HCWs screened negative. Acquisition of CNPA was associated with longer ICU stay (adjusted hazard ratio = 1.89, 99% confidence interval 1.12-3.13). Mortality was >40% among patients with CNPA versus <30% among those without CNPA (P = 0.019). Moreover, 83/119 (69.7%) CNPA carried either blaVIM (n = 36), blaIMP (n = 23) or blaGES-5 (n = 24). Four sequence types (STs) dominated (ST235, ST823, ST446 and ST357). Five major MLVA clusters were distinguished, two belonging to ST235 and the other three to ST823, ST446 and ST357. CNPA are introduced into these ICUs and some strains expand clonally among patients and the environment, creating endemic CNPA. VIM-, IMP- and GES-5 genes are prevalent. CNPA acquisition was associated with prolonged ICU stay and may affect ICU survival.

Phenotypic and Genomic Characterization of AmpC-Producing Klebsiella pneumoniae From Korea.

The prevalence of multidrug-resistant gram-negative bacteria has continuously increased over the past few years; bacterial strains producing AmpC ß-lactamases and/or extended-spectrum ß-lactamases (ESBLs) are of particular concern. We combined high-resolution whole genome sequencing and phenotypic data to elucidate the mechanisms of resistance to cephamycin and ß-lactamase in Korean Klebsiella pneumoniae strains, in which no AmpC-encoding genes were detected by PCR. We identified several genes that alone or in combination can potentially explain the resistance phenotype. We showed that different mechanisms could explain the resistance phenotype, emphasizing the limitations of the PCR and the importance of distinguishing closely-related gene variants.