Evolution of mutations in the ftsI gene leading to amino acid substitutions in PBP3 in Haemophilus influenzae strains under the selective pressure of ampicillin and cefuroxime.

BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to ß-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Enterobacterales and Pseudomonas aeruginosa.

The objective of this study was to assess the susceptibility of cefiderocol against multidrug-resistant carbapenemase-producing and nonproducing bacteria. The panel comprised 182 isolates of the order Enterobacterales, and 40 strains of Pseudomonas aeruginosa. Antimicrobial susceptibility testing has been performed using broth microdilution method according to the European Committee on Antimicrobial Susceptibility Testing recommendations. Mass spectrometry matrix-assisted laser desorption/ionization-time of flight mass spectrometry and carbapenemase-producing test were used to verify the presence of carbapenemases in clinical isolates. The genetic expression of single carbapenemases (blaKPC, blaOXA-48, blaNDM, blaVIM, blaIMP, blaGES) was determined by real-time polymerase chain reaction. Cefiderocol exhibited a good activity against the majority of strains tested in this study. Altogether, growth of 81.9% (n = 149) strains of the order Enterobacterales and 77.5% (n = 31) of P. aeruginosa isolates were inhibited at minimal inhibitory concentration (MIC) ≤2 mg/L. Values MIC50/MIC90 were 0.5/8 mg/L for enterobacteria, and 1/8 mg/L for P. aeruginosa. One isolate (Klebsiella pneumoniae) harboring two carbapenemases (blaOXA-48, blaNDM) had cefiderocol MIC 0.5 mg/L. In enterobacteria resistant to cefiderocol, blaNDM carbapenemase prevailed (43.3%, n = 29), followed by blaOXA-48 (31.3%, n = 21) and blaKPC (4.5%, n = 3). blaIMP (n = 8) and blaVIM (n = 1) metallo-ß-lactamases dominated in cefiderocol-resistant P. aeruginosa (n = 9) isolates. Very good susceptibility (100%) to this drug showed blaGES-positive strains of P. aeruginosa (n = 8) and isolates resistant to meropenem without confirmed carbapenemase gene (n = 10). In this study, cefiderocol demonstrated potent activity against important nosocomial pathogens, therefore, therapeutic options of this drug against multidrug-resistant bacteria should be considered.

[Case report of fatal sepsis caused by Actinobacillus suis/equuli in an adult male].

This is a case report of sepsis caused by the species Actinobacillus suis/equuli in a male agriculture worker that ended fatally. The article also contains information on identification and results of antibiotic susceptibility testing. This is a rare case of human infection and probably the first case of a human being infected by this species in the Czech Republic.

The first case of Planococcus glaciei found in blood, a report from the Czech Republic.

The isolation of Planococcus glaciei (designed strain CNCTC 7660) from blood of a patient with appendicitis is reported. Species P. glaciei (type strain CGMCC 1.6846 T) was for the first time identified as an environmental bacterium acquired from a glacier in China in 2009. To reveal the identity of the isolate CNCTC 7660, the 16S rDNA sequencing and the whole genome sequencing (Illumina MiSeq, Oxford Nanopore) were performed. The level of 16S rDNA gene sequencing similarity between CNCTC 7660 and CGMCC 1.6846 T was 99.55%. Phylogenetic analysis and average nucleotide analysis (ANI) based on the whole genome sequencing confirmed that the isolate CNCTC 7660 and CGMCC1.6846 T had ANI value above the taxonomic threshold for belonging to the same species (95%). The G + C content of CNCTC 7660 DNA was 46.8% (mol/mol). Except for the growth temperature, strains CGMCC1.6846 T and CNCTC 7660 were distinguished also biochemically. Due to the lack of information about the pathogenicity of P. glaciei, the possibility that it exerts pathogenicity in persons is suggested. But for understanding the nature of this species, further cases are needed.

Characterization of Haemophilus influenzae Strains with Non-Enzymatic Resistance to ß-Lactam Antibiotics Caused by Mutations in the PBP3 Gene in the Czech Republic in 2010-2018.

The surveillance data on antibiotic resistance of Haemophilus influenzae have shown that strains with non-enzymatic resistance to ß-lactam antibiotics have been on the rise in the Czech Republic over the last decade. This type of resistance is more difficult to detect than ß-lactamase production. Analysis of 228 H. influenzae strains revealed that isolates with non-enzymatic resistance to ß-lactams due to mutations in the ftsI gene could be reliably demonstrated by single run testing of susceptibility to amoxicillin/clavulanic acid (sensitivity of detection is 84.6%), cefuroxime (92.6%), ampicillin and penicillin (both 95.7%). Thirty-seven different amino acid substitution combinations were detected in the PBP3 protein at 23 positions (V329I, D350N, S357N, A368T, M377I, S385T, A388V, L389F, P393L, A437S, I449V, G490E, I491V, R501L, A502S, A502T, A502V, V511A, R517H, I519L, N526K, A530S, and T532S). The most common combination (35%) of amino acid substitutions was the combination D350N, M377I, A502V, N526K. Epidemiological typing does not indicate a clonal spread of a particular MLST type. Altogether there has been detected 74 STs. The most prevalent ST 1034 was associated mainly with a combination D350N, M377I, A502V, N526K. Clonal analysis revealed six clonal complexes (CCs) with the founder found, eight CCs without founder and 33 singletons.

Antibiotic Resistance, spa Typing and Clonal Analysis of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Blood of Patients Hospitalized in the Czech Republic.

Staphylococcus aureus is one of the major causes of bloodstream infections. The aim of our study was to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolates from blood of patients hospitalized in the Czech Republic between 2016 and 2018. All MRSA strains were tested for antibiotic susceptibility, analyzed by spa typing and clustered using a Based Upon Repeat Pattern (BURP) algorithm. The representative isolates of the four most common spa types and representative isolates of all spa clonal complexes were further typed by multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. The majority of MRSA strains were resistant to ciprofloxacin (94%), erythromycin (95.5%) and clindamycin (95.6%). Among the 618 strains analyzed, 52 different spa types were detected. BURP analysis divided them into six different clusters. The most common spa types were t003, t586, t014 and t002, all belonging to the CC5 (clonal complex). CC5 was the most abundant MLST CC of our study, comprising of 91.7% (n = 565) of spa-typeable isolates. Other CCs present in our study were CC398, CC22, CC8, CC45 and CC97. To our knowledge, this is the biggest nationwide study aimed at typing MRSA blood isolates from the Czech Republic.

Spread of Linezolid-Resistant Enterococcus spp. in Human Clinical Isolates in the Czech Republic.

The aim of this study was to map and investigate linezolid resistance mechanisms in linezolid-resistant enterococci in the Czech Republic from 2009 to 2019. Altogether, 1442 isolates of Enterococcus faecium and Enterococcus faecalis were examined in the National Reference Laboratory for Antibiotics. Among them, 8% of isolates (n = 115) were resistant to linezolid (E. faecium/n = 106, E. faecalis/n = 9). Only three strains of E. faecium were resistant to tigecycline, 72.6% of isolates were resistant to vancomycin. One isolate of E. faecium harbored the cfr gene. The majority (87%, n = 11) of E. faecium strains were resistant to linezolid because of the mutation G2576T in the domain V of the 23S rRNA. This mutation was detected also in two strains of E. faecalis. The presence of the optrA gene was the dominant mechanism of linezolid resistance in E. faecalis isolates. None of enterococci contained cfrB, poxtA genes, or any amino acid mutation in genes encoding ribosomal proteins. No mechanism of resistance was identified in 4 out of 106 E. faecium linezolid resistant isolates in this study. Seventeen sequence types (STs) including four novel STs were identified in this work. Clonal complex CC17 was found in all E. faecium isolates.

[Our experiences with Actinomyces urogenitalis in human clinical samples].

Actinomyces urogenitalis is most commonly associated with the human genitourinary system, often only as the resident flora. Outside the genitourinary tract, A. urogenitalis is isolated rather sporadically. Presented are two brief case reports of human infections outside the genitourinary tract as well as experiences with microbiological identification of this actinomycete. Antibiotic susceptibility testing of actinomycetes is focused especially on their resistance to lincosamides and fluoroquinolones. The etiological relationship with the patients' clinical problems was not investigated. Previously reported cases of infections outside the genitourinary tract are also mentioned in the article. The article may aid in expanding the knowledge of the occurrence, diagnosis and susceptibility of A. urogenitalis to antibiotics, particularly in rarely reported extra-genitourinary infections caused by this species. Accurate species identification in routine laboratory practice is important both for determination of the etiological role of the microorganism and for more precise selection of empirical antibiotic therapy.

Whole genome sequencing of macrolide resistant Streptococcus pneumoniae serotype 19A sequence type 416.

BACKGROUND: The resistance of Streptococcus pneumoniae to macrolides is becoming an increasingly important issue and thus it is important to understand the genetics related to adaptation of this species to the widespread use of antibiotics in Europe. The 58 isolates of S. pneumoniae belonging to sequence type (ST) 416 and serotype 19A and to several different phenotypes originated from Italy, Portugal and Czech Republic were thus sequenced on Illumina MiSeq. The aim of the study was to describe genetical origine of isolates, investigate their macrolide resistance and suggest reasons for spread of ST416 in the Czech Republic. RESULTS: Investigation of genes associated with serotype determined serotype switch between 15B and 19A serotypes and core genome multilocus sequence typing (cgMLST) confirmed the origine of concerned isolates in Netherlands15B-37 clone. Inspected genomes proved variability of genes associated with the macrolide resistance even within closely genetically relative isolates. CONCLUSIONS: Participation of 19A/ST416 on the spread of Netherlands15B-37 is accompanied by serotype switch between 19A and 15B serotypes and with acquisition of genes involved in macrolide resistance to the clone that was originally macrolide susceptible. There is evident tendency to interchanging and modifications of these and surrounding genes, that could lead to accelerate spreading of this sequence type in regions with high macrolide consumption.

Isolation of Bordetella trematum from the respiratory tract of a patient with lung cancer: a case report.

We report the case of isolation of Bordetella trematum from the respiratory tract of a patient with lung carcinoma. This gram-negative, opportunistic rod was firstly described in 1996. To date, only several strains of Bordetella trematum have been isolated and reported, mostly from skin and soft tissue infections. The patient was admitted to the ICU of the Pulmonary Department in incipient septic shock with respiratory failure. Intravenous fluid resuscitation and non-invasive ventilation were administered immediately. A broad spectrum antibiotic piperacillin/tazobactam was administered empirically after sampling of material for microbiological examination. The bronchoscopy showed a large cavern of decayed tumour invading into mediastinum. Both sample cultures showed significant quantities of gram-negative non-fermenting bacteria. The isolate was identified using MALDI-TOF MS as Bordetella trematum and the identification was confirmed using 16S ribosomal RNA sequencing. In the last few years, routine bacterial identification using MALDI-TOF MS has enabled correct discrimination of this species. Nevertheless, isolation of Bordetella trematum in clinical samples is still very uncommon, and it is appropriate to confirm the species identification via 16S ribosomal RNA sequencing. To our knowledge, this is the first case of B. trematum isolated from the human respiratory tract since its first description. The clinical significance of Bordetella trematum in the rapid deterioration of the patient's status remains unclear.

Phenotype and Genotype Characteristics of Staphylococcus aureus Resistant to Methicillin/Oxacillin Carrying Gene mecC in the Czech Republic from 2002 to 2017.

The aim of this study was to detect and characterize isolates of methicillin-/oxacillin-resistant Staphylococcus aureus (MRSA) carrying gene mecC (MRSA/mecC) and occurring in the Czech Republic within the period from 2002 to 2017. Altogether, 18 from 3,472 isolates of MRSA were mecC positive (0.52%). The first detection of MRSA/mecC in the Czech Republic is dated to 2004. MRSA/mecC isolates were susceptible to almost all tested antibiotics with few exceptions. Resistances to erythromycin (n = 2), clindamycin (n = 1), trimethoprim-sulfamethoxazole (n = 1), and rifampicin (n = 1) were found in the collection. Multilocus sequence typing and spa typing revealed a genetic heterogeneity of MRSA/mecC strains: three CCs (130, 425, and 2361), five STs (1245, 130, 2361, 425, and a new ST5480), and eight spa types (t843, t978, t1048, t1535, t1736, t6104, t8842, and t17153), which were detected in the study, with the highest prevalence of CC130/t843 lineage (n = 8, 44%). Except for two strains, none from 18 examined isolates harbored genes encoding any of S. aureus toxins: enterotoxins a-u, exfoliative toxins A, B, and D, toxic shock syndrome toxin-1, and the Panton-Valentine leukocidin.

[Unusual microbiological findings in bacteremia cases - three case reports].

The authors present three case reports of bacteremia with very rare microorganisms being isolated in the patients. Each brief case report is accompanied by etiological considerations related to the diagnosis and clinical condition of the patient. Routine microbiology tests were performed by examining the biochemical properties using commercial kits. Two methods were used to determine the microorganism species in the State Institute of Public Health in Prague: MALDI-TOF MS and 16S rDNA sequencing. Antibiotic susceptibility was assessed by the disk diffusion test; minimum inhibitory concentrations were determined using broth microdilution and the gradient diffusion method (E tests). This is probably the first reported case of Stenotrophomonas acidaminiphila isolated from human clinical specimens.

The association of pili with the emergence and replacement of the major antibiotic resistant pneumococcal clones.

BACKGROUND: The aim of this study was to evaluate the presence of pilus islet 1 (PI-1) and to determine its clade type in pneumococcal isolates with reduced susceptibility to penicillin (penicillin non-susceptible pneumococci - PNSP) and/or resistant to macrolides isolated prior to and after the introduction of pneumococcal conjugate vaccines (PCVs) in the Czech Republic. METHODS: Clinical isolates of serotypes 9V (n = 68) and 19A (n = 89) were examined. Isolates were characterised by multilocus sequence typing (MLST). The presence of PI-1 was determined by screening for the sortase B, C, and D genes located within PI-1. In the presence of PI-1 pilus, clade types were classified by PCR. RESULTS: In the pre-PCV period (2000-2007), the prevalence of PNSP was 3.9% and 2.7% of isolates were resistant to erythromycin. During 2012-2015 (post-PCV period), the rates of PNSP remained stable (3.6%), but resistance to erythromycin increased to 8.3%. While in 2000-2007, resistance to antibiotics was associated mainly with serotype 9V, in 2012-2015, it was replaced by serotype 19A. PI-1 positive isolates were seen in both serotypes. All isolates (68) of serotype 9V belonged to the Spain9V-3 (CC156) clone and carried PI-1 of clade type I while 96.5% (56/58) of isolates of 19A serotype belonged to the Netherlands15B-37 (CC199) clone and carried PI-1 of clade type II. CONCLUSIONS: Both major antibiotic resistant clones carried PI-1, although they differ in the clade type. Thus the role of PI-1 should be evaluated in further studies and potentially considered in the spread of antibiotic resistant clones.

Correct species identification (reclassification in CNCTC) of strains of Staphylococcus intermedius-group can improve an insight into their evolutionary history.

A group of 59 putative strains of Staphylococcus intermedius/Staphylococcus pseudintermedius deposited in the Czech National Collection of Type Cultures (CNCTC, National Institute for Public Health, Prague, Czech Republic) and the National Reference Laboratory for Staphylococci (NRL for Staphylococci, National Institute for Public Health, Prague, Czech Republic) was reclassified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There the biggest human collection of S. pseudintermedius in Europe was analysed; 44 samples (75%) were of human origin. Twenty-two percent (n = 13) of the strains were isolated from animals, and two staphylococci were of unknown origin. This study revealed the prevalence of Staphylococcus pseudintermedius (94%, n = 53) vs. Staphylococcus intermedius (6%, n = 6) in the collection of human and veterinary staphylococci after reclassification. Results of PCR-RFLP analysis were verified by comparison with a repetitive element sequence-based polymerase chain reaction (Rep-PCR) analysis on 26 (44%) randomly selected strains. Due to a low-resolution ability of PCR-RFLP to separate Staphylococcus intermedius from Staphylococcus delphini, four isolates of Staphylococcus intermedius were biochemically verified further to exclude the presence of Staphylococcus delphini in the collection. Our results indicate that S. intermedius and S. pseudintermedius have occurred independently over an age-long period of their co-evolution.

Molecular characterization of serogroup 19 Streptococcus pneumoniae in the Czech Republic in the post-vaccine era.

Purpose. The aim of this study was to characterize serogroup 19 isolates resistant to macrolides and/or penicillin found among pneumococci recovered from cases of invasive and respiratory tract disease in the Czech Republic in 2014.Methods. Pneumococcal isolates of serotypes 19A (n=26) and 19F (n=10) that were non-susceptible to penicillin and/or macrolides and had been collected in 2014 were analysed using multi-locus sequence typing (MLST). Four isolates representing the major clones were subjected to whole-genome sequencing (WGS).Results. The penicillin-susceptible macrolide-resistant isolates of serotype 19A were mainly associated with sequence type (ST) 416 belonging to clonal complex (CC) 199, and the penicillin-resistant isolates were of serotype 19F belonging to ST1464 (CC 320). WGS revealed the presence of pilus 1, in association with pilus 2, in serotype19F isolates belonging to CC 320. Another adhesin, pneumococcal serine-rich protein (PsrP), was only present in serotype 19A isolates of ST416. Analysis of the penicillin-binding proteins (PBPs) of serotype 19F penicillin-resistant isolates (ST1464 and ST271) performed on PBP1a, 2b and 2x identified a large number of mutations in comparison to the reference strain, R6. Both isolates contained a unique PBP profile; however, they were highly similar to PBP sequences of the Taiwan19F-14 reference strain. The Pbp2b sequences of both 19F isolates showed the lowest similarity to those of the Taiwan19F-14 strain (91 % similarity), while they were also found to be distantly related to each other (94 % similarity).Conclusions. WGS revealed specific virulence factors in antibiotic-resistant pneumococcal clones that spread rapidly in the post-vaccine era in the Czech Republic.

Acute hyperlipidemia initiates proinflammatory and proatherogenic changes in circulation and adipose tissue in obese women.

BACKGROUND: Obesity represents a high risk factor for the development of atherosclerosis and is associated with a low-grade inflammation and activation of immune cells. AIMS: The aim of our study was to investigate the effect of a short-term lipid infusion on immune cells in blood and subcutaneous abdominal adipose tissue (SAAT) in obese women. METHODS: Seven-hour intravenous lipid/control infusions were performed in two groups of women (n = 15, n = 10, respectively). Before and at the end of the infusion, SAAT and blood samples were obtained and relative content and phenotype of immune cells were analyzed using flow cytometry. Analysis of immune cell markers, inflammation and angiogenesis markers was performed in SAAT by RT-PCR and in plasma by immunoassays. RESULTS: Relative content of CD45+/14+ and CD45+/14+/16+ populations of monocytes was reduced in circulation by 21% (p = 0.004) and by 46% (p = 0.0002), respectively, in response to hyperlipidemia, which suggested the increased adhesion of these cells to endothelium. In line with this, the levels of sICAM and sVCAM in plasma were increased by 9.4% (p = 0.016), 11.8% (p = 0.008), respectively. In SAAT, the relative content of M2 monocyte/macrophages subpopulation CD45+/14+/206+/16+ decreased by 27% (p = 0.012) and subpopulations CD14+/CD206- and CD14/+TLR4+ cells increased (p = 0.026; p = 0.049, respectively). Intralipid infusion promoted an increase of mRNA levels in SAAT: RORC (marker of proinflammatory Th17 lymphocytes) by 43% (p = 0.048), MCP-1 (78%, p = 0.028) and VEGF (68.5%, p = 0.0001). CONCLUSIONS: Acute hyperlipidemia induces a proinflammatory and proatherogenic response associated with altered relative content of immune cells in blood and SAAT in obese women.

Protective Effect of Unsaturated Fatty Acids on Palmitic Acid-Induced Toxicity in Skeletal Muscle Cells is not Mediated by PPARδ Activation.

Unsaturated free fatty acids (FFA) are able to prevent deleterious effects of saturated FFA in skeletal muscle cells although the mechanisms involved are still not completely understood. FFA act as endogenous ligands of peroxisome proliferator-activated receptors (PPAR), transcription factors regulating the expression of genes involved in lipid metabolism. The aim of this study was to determine whether activation of PPARδ, the most common PPAR subtype in skeletal muscle, plays a role in mediating the protective effect of unsaturated FFA on saturated FFA-induced damage in skeletal muscle cells and to examine an impact on mitochondrial respiration. Mouse C2C12 myotubes were treated for 24 h with different concentrations of saturated FFA (palmitic acid), unsaturated FFA (oleic, linoleic and α-linolenic acid), and their combinations. PPARδ agonist GW501516 and antagonist GSK0660 were also used. Both mono- and polyunsaturated FFA, but not GW501516, prevented palmitic acid-induced cell death. Mono- and polyunsaturated FFA proved to be effective activators of PPARδ compared to saturated palmitic acid; however, in combination with palmitic acid their effect on PPARδ activation was blocked and stayed at the levels observed for palmitic acid alone. Unsaturated FFA at moderate physiological concentrations as well as GW501516, but not palmitic acid, mildly uncoupled mitochondrial respiration. Our results indicate that although unsaturated FFA are effective activators of PPARδ, their protective effect on palmitic acid-induced toxicity is not mediated by PPARδ activation and subsequent induction of lipid regulatory genes in skeletal muscle cells. Other mechanisms, such as mitochondrial uncoupling, may underlie their effect.

Experimental hyperglycemia induces an increase of monocyte and T-lymphocyte content in adipose tissue of healthy obese women.

BACKGROUND/OBJECTIVES: Hyperglycemia represents one of possible mediators for activation of immune system and may contribute to worsening of inflammatory state associated with obesity. The aim of our study was to investigate the effect of a short-term hyperglycemia (HG) on the phenotype and relative content of immune cells in circulation and subcutaneous abdominal adipose tissue (SAAT) in obese women without metabolic complications. SUBJECTS/METHODS: Three hour HG clamp with infusion of octreotide and control investigations with infusion of octreotide or saline were performed in three groups of obese women (Group1: HG, Group 2: Octreotide, Group 3: Saline, n=10 per group). Before and at the end of the interventions, samples of SAAT and blood were obtained. The relative content of immune cells in blood and SAAT was determined by flow cytometry. Gene expression analysis of immunity-related markers in SAAT was performed by quantitative real-time PCR. RESULTS: In blood, no changes in analysed immune cell population were observed in response to HG. In SAAT, HG induced an increase in the content of CD206 negative monocytes/macrophages (p<0.05) and T lymphocytes (both T helper and T cytotoxic lymphocytes, p<0.01). Further, HG promoted an increase of mRNA levels of immune response markers (CCL2, TLR4, TNFα) and lymphocyte markers (CD3g, CD4, CD8a, TBX21, GATA3, FoxP3) in SAAT (p<0.05 and 0.01). Under both control infusions, none of these changes were observed. CONCLUSIONS: Acute HG significantly increased the content of monocytes and lymphocytes in SAAT of healthy obese women. This result suggests that the short-term HG can modulate an immune status of AT in obese subjects.