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.

Phenotypic and Genomic Variability of Serial Peri-Lung Transplantation Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients.

Cystic fibrosis (CF) represents one of the major genetic and chronic lung diseases affecting Caucasians of European descent. Patients with CF suffer from recurring infections that lead to further damage of the lungs. Pulmonary infection due to Pseudomonas aeruginosa is most prevalent, further increasing CF-related mortality. The present study describes the phenotypic and genotypic variations among 36 P. aeruginosa isolates obtained serially from a non-CF and five CF patients before, during and after lung transplantation (LTx). The classical and genomic investigation of these isolates revealed a common mucoid phenotype and only subtle differences in the genomes. Isolates originating from an individual patient shared ≥98.7% average nucleotide identity (ANI). However, when considering isolates from different patients, substantial variations in terms of sequence type (ST), virulence factors and antimicrobial resistance (AMR) genes were observed. Whole genome multi-locus sequence typing (MLST) confirmed the presence of unique STs per patient regardless of the time from LTx. It was supported by the monophyletic clustering found in the genome-wide phylogeny. The antibiogram shows that ≥91.6% of the isolates were susceptible to amikacin, colistin and tobramycin. For other antibiotics from the panel, isolates frequently showed resistance. Alternatively, a comparative analysis of the 36 P. aeruginosa isolates with 672 strains isolated from diverse ecologies demonstrated clustering of the CF isolates according to the LTx patients from whom they were isolated. We observed that despite LTx and associated measures, all patients remained persistently colonized with similar isolates. The present study shows how whole genome sequencing (WGS) along with phenotypic analysis can help us understand the evolution of P. aeruginosa over time especially its antibiotic resistance.

Whole-genome sequencing of Egyptian multidrug-resistant Klebsiella pneumoniae isolates: a multi-center pilot study.

Multidrug-resistant (MDR) Klebsiella pneumoniae is a common infectious pathogen. We performed whole-genome sequencing (WGS) of 39 randomly selected, geographically diverse MDR K. pneumoniae from nine Egyptian hospitals. Clinical sources, phenotypic antibiotic resistance, and hyper-mucoviscosity were documented. WGS data were epidemiologically interpreted and tested for the presence of antibiotic resistance and virulence genes. Based on WGS data, we identified 18 classical multi-locus sequence types (MLST), the most common type being ST101 (23.1%) followed by ST147 (17.9%). Phylogenetic analyses identified small numbers of closely related isolates in a few of the centers, so we mostly documented independent nosocomial acquisition or import from public sources. The most common acquired resistance gene found was blaCTX-M-15, detected in 27 isolates (69.2%). Carbapenemase genes encountered were blaNDM-1 (n = 13), blaNDM-5 (n = 1), blaOXA-48 (n = 12), blaOXA-181 (n = 2), and blaKPC2 (n = 1). Seven strains (18%) contained more than a single carbapenemase gene. While searching for virulence-associated genes, sixteen wzi alleles were identified with wzi137, wzi64, and wzi50 most commonly found in ST101, ST147, and ST16, respectively. Yersiniabactin was the most common virulence factor (69.2%). Hyper-mucoviscosity was documented for 6 out of 39 isolates.This is the first genomic study of MDR K. pneumoniae from Egypt. The study revealed a clear spread of well-known international clones and their associated antimicrobial resistance and (hyper)virulence traits. The clinical situation in Egypt seems to reflect the scenario documented in many other countries and requires close attention.

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.

Discrimination of Escherichia coli and Shigella spp. by Nuclear Magnetic Resonance Based Metabolomic Characterization of Culture Media.

Dysentery is a major health threat that dramatically impacts childhood morbidity and mortality in developing countries. Various pathogenic agents cause dysentery, such as Shigella spp. and Escherichia coli, which are very closely related if not identical species. Sensitive and precise detection and identification of the infectious agent is important to target the best therapeutic strategy, but the differential diagnosis of these two groups remains a challenge using conventional methods. Here, we present a nuclear magnetic resonance (NMR) based multivariate classification model employing bacterial metabolic footprints in postculture growth media with remarkable segregation capability, including the discrimination of lactose negative E. coli and Shigella spp. Our results confirm the potential of metabolomic markers in the field of bacterial identification for the distinction of even very closely related species.

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.

Genomic Evolution of Staphylococcus aureus During Artificial and Natural Colonization of the Human Nose.

Staphylococcus aureus can colonize the human vestibulum nasi for many years. It is unknown whether and, how S. aureus adapts to this ecological niche during colonization. We determined the short (1 and 3 months) and mid-term (36 months) genomic evolution of S. aureus in natural carriers and artificially colonized volunteers. Eighty-five S. aureus strains were collected from 6 natural carriers during 3 years and 6 artificially colonized volunteers during 1 month. Multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis based on whole-genome sequencing (WGS) were carried out. Mutation frequencies within resident bacterial populations over time were quantified using core genome SNP counts (comparing groups of genomes) and pairwise SNP divergence assessment (comparing two genomes from strains originating from one host and sharing identical MLST). SNP counts (within 1-3 months) in all naturally colonizing strains varied from 0 to 757 (median 4). These strains showed random and independent patterns of pairwise SNP divergence (0 to 44 SNPs, median 7). When the different core genome SNP counts over a period of 3 years were considered, the median SNP count was 4 (range 0-26). Host-specific pairwise SNP divergence for the same period ranged from 9 to 57 SNPs (median 20). During short term artificial colonization the mutation frequency was even lower (0-7 SNPs, median 2) and the pairwise SNP distances were 0 to 5 SNPs (median 2). Quantifying mutation frequencies is important for the longitudinal follow-up of epidemics of infections and outbreak management. Random pattern of pairwise SNP divergence between the strains isolated from single carriers suggested that the WGS of multiple colonies is necessary in this context. Over periods up to 3 years, maximum median core genome SNP counts and SNP divergence for the strains studied were 4 and 20 SNPs or lower. During artificial colonization, where median core genome SNP and pairwise SNP distance scores were 2, there is no early stage selection of different genotypes. Therefore, we suggest an epidemiological cut off value of 20 SNPs as a marker of S. aureus strain identity during studies on nasal colonization and also outbreaks of infection.

Genomic evolution and local epidemiology of Klebsiella pneumoniae from a major hospital in Beijing, China, over a 15 year period: dissemination of known and novel high-risk clones.

Klebsiella pneumoniae is a frequent cause of nosocomial and severe community-acquired infections. Multidrug-resistant (MDR) and hypervirulent (hv) strains represent major threats, and tracking their emergence, evolution and the emerging convergence of MDR and hv traits is of major importance. We employed whole-genome sequencing (WGS) to study the evolution and epidemiology of a large longitudinal collection of clinical K. pneumoniae isolates from the H301 hospital in Beijing, China. Overall, the population was highly diverse, although some clones were predominant. Strains belonging to clonal group (CG) 258 were dominant, and represented the majority of carbapenemase-producers. While CG258 strains showed high diversity, one clone, ST11-KL47, represented the majority of isolates, and was highly associated with the KPC-2 carbapenemase and several virulence factors, including a virulence plasmid. The second dominant clone was CG23, which is the major hv clone globally. While it is usually susceptible to multiple antibiotics, we found some isolates harbouring MDR plasmids encoding for ESBLs and carbapenemases. We also reported the local emergence of a recently described high-risk clone, ST383. Conversely to strains belonging to CG258, which are usually associated to KPC-2, ST383 strains seem to readily acquire carbapenemases of different types. Moreover, we found several ST383 strains carrying the hypervirulence plasmid. Overall, we detected about 5 % of simultaneous carriage of AMR genes (ESBLs or carbapenemases) and hypervirulence genes. Tracking the emergence and evolution of such strains, causing severe infections with limited treatment options, is fundamental in order to understand their origin and evolution and to limit their spread. This article contains data hosted by Microreact.

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.

A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis.

The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student's t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol.

In vitro efficiency of the piperacillin/tazobactam combination against inhibitor-resistant TEM- and complex mutant TEM-producing clinical strains of Escherichia coli.

OBJECTIVES: We investigated the bacteriostatic and bactericidal activities of piperacillin/tazobactam against 16 clinical Escherichia coli producing inhibitor-resistant TEM ß-lactamases (IRT; 13/16) and complex mutant TEM enzymes (CMT; 3/16). METHODS: Bacteriostatic activity was evaluated by three methods (disc diffusion, Vitek2 automated system, MIC determination by a microdilution method) and a time-killing study was used to investigate the bactericidal effect against standard (5 × 10(5) cfu/mL) and high inocula (5 × 10(6) cfu/mL). RESULTS: Piperacillin/tazobactam was bacteriostatic against most of the tested strains (15/16). Using a high inoculum, the piperacillin/tazobactam combination was not bactericidal against the 13 IRT-producing strains and one of the CMT-producing strains (1/3). A loss of bactericidal activity was still observed for seven IRT-producing strains (7/13) with a standard bacterial inoculum (<99.9% killing over 24 h). CONCLUSIONS: Despite usual in vitro bacteriostatic activity, the piperacillin/tazobactam combination was not bactericidal against most IRT-producing clinical strains of E. coli, especially for the treatment of a high bacterial inoculum. This possible loss of bactericidal effect should be brought to the attention of physicians and may require high dosing regimens for the treatment of severe infections.

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A.

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K (m) and V (max) of 1.6 mg ml(-1) and 118 micromol min(-1) mg(-1) on oat spelt xylan, and its optimal temperature and pH for activity were 37 degrees C and pH 6.0, respectively. Its catalytic properties (k (cat)/K (m) = 3,300 ml mg(-1) min(-1)) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.