Structure of a laminarin-type ß-(1→3)-glucan from brown algae Sargassum henslowianum and its potential on regulating gut microbiota.

A homogeneous polysaccharide named SHNP with apparent molecular weight of 8.4 kDa was purified from brown algae Sargassum henslowianum using ethanol precipitation, ion-exchange chromatography, and gel-filtration column chromatography. Structural analyses reveal that SHNP is completely composed of glucose, and its backbone consists of ß-D-(1→3)-Glcp with side chains comprising t-ß-D-Glcp attached at the O-6 position. Thus, SHNP is a laminarin-type polysaccharide. In vitro fermentation test results showed that SHNP was digested by gut microbiota; the pH value in the fecal culture of SHNP was significantly decreased; and total short-chain fatty acids, acetic, propionic and n-butyric acids were significantly increased. Furthermore, SHNP regulated the intestinal microbiota composition by stimulating the growth of species belonging to Enterobacteriaceae while depleting Haemophilus parainfluenzae and Gemmiger formicilis. Taken together, these results indicate that SHNP has the potential for regulating gut microbiota, but its specific role in the regulation requires to be further investigated.

Prevalence of susceptibility patterns of opportunistic bacteria in line with CLSI or EUCAST among Haemophilus parainfluenzae isolated from respiratory microbiota.

The application of CLSI and EUCAST guidelines led to many discrepancies. Various doubts have already appeared in preliminary stages of microbiological diagnostics of Haemophilus spp. A total of 87 H. parainfluenzae isolates were obtained from throat or nasopharyngeal swabs from adults 18 to 70 years old, both healthy volunteers and patients with chronic diseases between 2013 to 2015 in eastern Poland. Haemophilus spp. were identified by colony morphology, Gram-staining, API NH and MALDI-TOF MS technique. Both susceptibility to various antimicrobials and phenotypes of Haemophilus spp. resistance to beta-lactams were determined. Statistically significant association between applied guidelines and drug resistance patterns were observed to as follows: ampicillin, cefuroxime, cefotaxime, amoxicillin-clavulanate, azithromycin, tetracycline and trimethoprim-sulfamethoxazole. Resistance phenotypes according to CLSI vs. EUCAST were as follows: 3.4% vs. 8.0% for BLNAR and 6.9% vs. 19.5% for BLPACR isolates. In conclusion, this is the first study that reports comparative analysis of drug susceptibility interpretation using CLSI and EUCAST of haemophili rods from human respiratory microbiota in Poland. In case of susceptible, increased exposure (formerly intermediate) category of susceptibility within H. parainfluenzae isolates we have observed EUCAST as more restrictive than CLSI. Moreover, BLNAI and BLPAI phenotype isolates have been observed, as well as BLPBR using only CLSI or EUCAST guidelines, respectively.

Emergence of multidrug resistance among Haemophilus parainfluenzae from respiratory and urogenital samples in Barcelona, Spain.

Haemophilus parainfluenzae (HPAR) is a Gram-negative bacterium that can become an opportunistic urogenital pathogen. Recently, multidrug resistant (MDR) strains have emerged. We aim to analyse the epidemiology of HPAR at Hospital Universitari de Bellvitge between 2013 and 2017 to determine its putative role in sexually transmitted infections (STI). Strains were classified by sample origin, and antimicrobial susceptibility was performed by disk-diffusion tested on Mueller-Hinton Fastidious. MDR was defined as the resistance of the antimicrobial to three or more antibiotic class. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) after restriction with SmaI and Cfr9I. We classified 944 HPAR isolates as being of urogenital (n = 175; 18.5%), respiratory (n = 719; 76.2%), or other (n = 50; 5.3%) origins. Among the urogenital isolates, 50 (28.6%) were MDR, which was significantly higher than that found in respiratory samples (40/719; 5.6%; p < 0.01). The frequency of MDR increased progressively among urogenital samples from 13.3% (2013) to 33.3% (2017) (r = 0.8; p = 0.035). The resistance rates for all 944 episodes were significantly higher for cotrimoxazole (51.4%), tetracycline (46.3%), chloramphenicol (28.0%), ciprofloxacin (21.1%), and ampicillin (20.6%). After PFGE, no clonal relationship was found. Clinical charts were available for 40 symptomatic patients with MDR HPAR infections presenting mostly urethritis (n = 26; 65.0%). In all cases, symptoms were treated effectively with combination therapy. Furthermore, in 10 of those patients with urethritis, MDR HPAR was the only potential pathogen to be identified. The emergence of MDR HPAR is a matter of concern, and the detection as a single pathogen highlights its putative role as cause of STI.

Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.

OBJECTIVES: To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. METHODS: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. RESULTS: Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. CONCLUSIONS: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.

Variability in nitrate-reducing oral bacteria and nitric oxide metabolites in biological fluids following dietary nitrate administration: An assessment of the critical difference.

There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CVB), and analytical imprecision (CVA) in experimental endpoints. However, no study has quantified the CVA and CVB of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CVB, CVA, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CVB of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CVB in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ±â€¯5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (∼12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitrate-reducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CVB for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CVB for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CVB for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CVB in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.

Antimicrobial activity of ceftaroline and comparator agents tested against organisms isolated from patients with community-acquired bacterial pneumonia in Europe, Asia, and Latin America.

OBJECTIVE: To evaluate the potency and spectrum of ceftaroline and comparator agents tested against contemporary bacteria isolated from patients with community-acquired bacterial pneumonia (CABP) in Europe (EUR), Asia-Pacific (APAC), and Latin America (LATAM). METHODS: A total of 4321 bacterial isolates were collected consecutively by the SENTRY Antimicrobial Surveillance Program in 2015-2017 from 65 medical centers located in Western Europe (W-EUR; 21 centers in 10 nations), Eastern Europe and the Mediterranean region (E-EUR; 15 centers in 11 nations), APAC (18 centers in nine nations), and LATAM (11 centers in nine nations). Isolates were collected from lower respiratory tract specimens, and an isolate obtained from an outpatient or earlier than 48h after hospitalization was considered community-acquired. Organisms were tested for susceptibility by reference broth microdilution methods in a central laboratory. RESULTS: Among Streptococcus pneumoniae (n=1736), 99.9% of isolates were ceftaroline-susceptible (MIC50/90, 0.008/0.12mg/l), and ceftriaxone susceptibility (≤1mg/l) ranged from 97.4% in W-EUR to 85.3% in the APAC region. Ceftaroline was also active against Haemophilus influenzae (n=1172; MIC50/90, 0.008/0.03mg/l; 99.8%/93.9% susceptible per CLSI/EUCAST criteria) and Staphylococcus aureus (n=777; MIC50/90, 0.25/1mg/l; 97.4% susceptible). Oxacillin resistance ranged from 31.9% in the APAC region to 15.0% in E-EUR. Ceftaroline also demonstrated potent activity against Moraxella catarrhalis (n=613; MIC50/90, 0.06/0.25mg/l) and Haemophilus parainfluenzae (n=23; MIC50/90, 0.015/0.03mg/l). CONCLUSIONS: Susceptibility rates varied widely by geographic region. Ceftaroline was active against the vast majority of bacterial organisms isolated from patients with CABP.

Substitutions in PBP3 confer resistance to both ampicillin and extended-spectrum cephalosporins in Haemophilus parainfluenzae as revealed by site-directed mutagenesis and gene recombinants.

Objectives: To determine the association of amino acid substitutions in PBP3 with ß-lactam susceptibility in Haemophilus parainfluenzae. Methods: Single and multiple amino acid mutations at positions 385, 511 and 526 were introduced into PBP3 of a ß-lactam-susceptible H. parainfluenzae strain using site-directed mutagenesis. Recombinants were also generated using PCR-amplified ftsI from clinical strains encoding multiple amino acid substitutions. MICs of ampicillin, cefuroxime, cefotaxime and ceftriaxone were determined using Etest®. Results: Transformation of a susceptible strain with ftsI from clinical strains encoding four substitutions in the transpeptidase region of PBP3 conferred resistance to ampicillin, but not to cephalosporins. Introduction of ftsI from a clinical strain encoding eight substitutions conferred resistance to ampicillin, cefotaxime and ceftriaxone. MICs for recombinants were lower than those for the donor strains. Using site-directed mutagenesis, no single substitution conferred resistance to the tested ß-lactams, although V511A increased the MIC of cefuroxime to the intermediate category for intravenous administration. Recombinants encoding N526K/H/S in combination with V511A were resistant to ampicillin. Substitution S385T increased the MICs of third-generation cephalosporins if V511A was also present. Conclusions: Substitutions in PBP3 are sufficient to confer resistance to both ampicillin and third-generation cephalosporins in H. parainfluenzae. A combination of substitutions at positions Val-511 and Asn-526 confers resistance to ampicillin. Resistance to third-generation cephalosporins probably requires more than four substitutions in PBP3.

Molecular Characterization of Fluoroquinolone-Resistant Haemophilus parainfluenzae Clinical Isolates in Spain.

OBJECTIVE: The objective was to characterize a group of clinical isolates of fluoroquinolone-resistant Haemophilus parainfluenzae collected in Northern Spain (March-December 2014). METHODS: Twelve clinical isolates of H. parainfluenzae were studied by performing antimicrobial susceptibility testing and PCR amplification and nucleotide sequencing of the QRDR (quinolone resistance-determining region) of gyrA, parC, gyrB, and parE genes. Screening for plasmid-mediated quinolone resistance (PMQR) was also studied. Pulsed-field gel electrophoresis (PFGE) was used for molecular typing. RESULTS: Antimicrobial susceptibility testing showed that all the isolates were resistant to the fluoroquinolones tested (ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin). Analysis of the QRDR demonstrated that all the isolates presented mutations in gyrA and parC. A Glu88Lys substitution in ParC is reported for the first time in H. parainfluenzae. No PMQR gene was detected. PFGE results showed that isolates were not clonally related. CONCLUSION: Multiple H. parainfluenzae fluoroquinolone-resistant isolates grouped in the same area in a short period of time showed diverse substitutions in QRDR of gyrA/parC and were not clonally related, indicating individual emergence. In addition, we described the first report of Glu88Lys substitution in ParC.

Fluoroquinolone, Macrolide, and Ketolide Resistance in Haemophilus parainfluenzae from South Africa.

Fluoroquinolones and ketolides are among the drugs of choice for the treatment of Haemophilus parainfluenzae infections. There has been a report of an emerging fluoroquinolone and telithromycin resistance in H. parainfluenzae isolates from the private sector of KwaZulu-Natal Province of South Africa that necessitates molecular investigation. The aim of this study is to characterize these resistance delineating mutations in genes commonly associated with reduced susceptibility. Ten H. parainfluenzae isolates retrieved from the sputum of 10 patients with H. parainfluenzae pneumonia were subjected to sensitivity testing by the disc diffusion and CLSI broth microdilution methods, polymerase chain reaction (PCR) and DNA sequencing of selected genes associated with resistance were carried out, while repetitive extragenic palindromic PCR (REP-PCR) was used to ascertain clonality. Fluoroquinolone resistance was attributed to the following amino acid substitutions: S84F, D88Y in GyrA, and S84Y/L, S138T, and M198 L change in ParC of the isolates. The plasmid-mediated quinolone resistance gene aac-(6')-Ib-cr was detected for the first time in four isolates of H. parainfluenzae and D420 N change was observed in ParE in one isolate. Macrolide and ketolide resistance were ascribed to the resistance genes mef (A), msr (D), and erm (B) detected in the isolates. REP-PCR analysis showed that the isolates were not clonal. All the observed resistance mechanisms are first reports in Africa. There is an emerging fluoroquinolone and macrolide resistance in H. parainfluenzae in South Africa that is attributable to known/novel resistance mechanisms, necessitating the monitoring of this pathogen as a potential opportunistic pathogen in a country with a high HIV and AIDS prevalence.

Molecular characterization of a clinical Haemophilus parainfluenzae isolate with cefotaxime resistance and decreased susceptibility to fluoroquinolones.

We report an H. parainfluenzae clinical isolate resistant to cefotaxime and with decreased susceptibility to ciprofloxacin recovered from a patient with cystic fibrosis. The isolate had elevated MICs of ampicillin (256mg/L), amoxicillin-clavulanate (8mg/L), cefuroxime (8mg/L) and cefotaxime (4mg/L), and showed a ß-lactamase-producing amoxicillin-clavulanic acid-resistant (BLPACR) phenotype. A blaTEM-1 plus five amino acid substitutions in the PBP3 were found: Ser385Thr, Val511Ala, Ile519Val, Asn526Lys and Asp551Leu. MIC of ciprofloxacin was 0.5mg/L, and substitutions in gyrA (Ser84Tyr) and parC (Ser84Phe) genes were detected.

Inhibitory effect of 1,2,4-triazole-ciprofloxacin hybrids on Haemophilus parainfluenzae and Haemophilus influenzae biofilm formation in vitro under stationary conditions.

Haemophilus parainfluenzae and Haemophilus influenzae, upper respiratory tract microbiota representatives, are able to colonize natural and artificial surfaces as biofilm. The aim of the present study was to assay the effect of ten 1,2,4-triazole-ciprofloxacin hybrids on planktonic or biofilm-forming haemophili cells in vitro under stationary conditions on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The reference strains of H. parainfluenzae and H. influenzae were included. The broth microdilution microtiter plate (MTP) method with twofold dilution of the compounds, or ciprofloxacin (reference agent) in 96-well polystyrene microplates, was used. The optical density (OD) reading was made spectrophotometrically at a wavelength of 570 nm (OD570) both to measure bacterial growth and to detect biofilm-forming cells under the same conditions with 0.1% crystal violet. The following values of parameters were estimated for 1,2,4-triazole-ciprofloxacin hybrids - MIC = 0.03-15.63 mg/L, MBIC = 0.03-15.63 mg/L, MBIC/MIC = 0.125-8, depending on the compound, and for ciprofloxacin - MIC = 0.03-0.06 mg/L, MBIC = 0.03-0.12 mg/L, MBIC/MIC = 1-2. The observed strong anti-adhesive properties (95-100% inhibition) of the tested compounds were reversible during long-term incubation at subinhibitory concentrations. Thus, 1,2,4-triazole-ciprofloxacin hybrids may be considered as starting compounds for designing improved agents not only against planktonic but also against biofilm-forming Haemophilus spp. cells.

Haemophilus parainfluenzae as a marker of the upper respiratory tract microbiota changes under the influence of preoperative prophylaxis with or without postoperative treatment in patients with lung cancer.

BACKGROUND: Haemophili are representative microbiota of the upper respiratory tract. The aim of this study was to assess the effects of perioperative antimicrobial prophylaxis and/or postoperative treatment on Haemophilus parainfluenzae prevalence, and antimicrobial sensitivity in short-term hospitalized patients with lung cancer who underwent surgery. RESULTS: Samples were collected from 30 short-term hospitalized patients with lung cancer and from 65 healthy people. The nasal and throat specimens were taken twice from each patient: before (EI, Examination I), on the fourth/fifth day (EII, Examination II) after surgery, and once from healthy people. The isolates identification and antimicrobial susceptibility were detected by routine diagnostic methods. H. parainfluenzae was found in throat specimens of 42/65 (64.6 %) healthy people, while in 19/30 (63.3 %) lung cancer patients in EI (p = 0.6203) and in 13/30 (43.3 %) ones in EII (p = 0.0106). Neither the disease itself nor short-term hospitalization with perioperative prophylaxis alone affected H. parainfluenzae prevalence in EII, while perioperative prophylaxis with postoperative treatment significantly decreased its colonization in EII. The differences in the number of patients colonized by Candida spp. in EI and in EII were observed (p = 0.0082).Totally, 23/58 (39.7 %) of H. parainfluenzae isolates were resistant mainly to beta-lactams; among 11 ampicillin-resistant isolates only 3 were beta-lactamase positive. CONCLUSIONS: The antimicrobial perioperative prophylaxis together with postoperative treatment may disturb the composition of the airways microbiota represented by H. parainfluenzae, in addition to selecting the resistant strains of bacteria and promoting yeasts colonization in lung cancer patients undergoing surgery.

Contribution of PBP3 Substitutions and TEM-1, TEM-15, and ROB-1 Beta-Lactamases to Cefotaxime Resistance in Haemophilus influenzae and Haemophilus parainfluenzae.

AIMS: To investigate the relative contributions of naturally occurring penicillin-binding protein 3 (PBP3) substitutions, and TEM-1, TEM-15, and ROB-1 beta-lactamases on resistance to a third-generation cephalosporin in Haemophilus influenzae and Haemophilus parainfluenzae. RESULTS: The minimum inhibitory concentration (MIC) of cefotaxime (CTX) was assessed after transformation with PCR-amplified ftsI genes expressing altered PBP3 and/or small plasmids encoding beta-lactamases into an isogenic environment of H. influenzae and H. parainfluenzae. Group III PBP3, comprising substitutions N526K, S385T, and L389F, conferred CTX resistance to H. influenzae according to EUCAST interpretative criteria. Group III-like PBP3, comprising substitutions N526H and S385T, increased the CTX MIC of H. parainfluenzae ninefold, but the level did not transgress the resistance breakpoint. Production of TEM-15 beta-lactamase conferred CTX resistance on both H. influenzae and H. parainfluenzae. A nitrocefin hydrolysis assay showed TEM-15 to be a less efficient enzyme compared to TEM-1. CONCLUSIONS: TEM-15 and PBP3 substitutions impose an additive effect on resistance to third-generation cephalosporins in both H. influenzae and H. parainfluenzae. The effect of PBP3 substitutions on beta-lactam resistance in H. parainfluenzae can be addressed by transfer of ftsI genes in vitro.

Emergence of extensively drug-resistant Haemophilus parainfluenzae in Switzerland.

Two homosexual men were colonized in the urethra with Haemophilus parainfluenzae nonsusceptible to ampicillin (MIC, 8 µg/ml), amoxicillin-clavulanate (MIC, 4 µg/ml), cefotaxime (MIC, 1.5 µg/ml), cefepime (MIC, 3 µg/ml), meropenem (MIC, 0.5 µg/ml), cefuroxime, azithromycin, ciprofloxacin, tetracycline, and chloramphenicol (all MICs, ≥ 32 µg/ml). Repetitive extragenic palindromic PCR (rep-PCR) showed that the strains were indistinguishable. The isolates had amino acid substitutions in PBP3, L4, GyrA, and ParC and possessed Mef(A), Tet(M), and CatS resistance mechanisms. This is the first report of extensively drug-resistant (XDR) H. parainfluenzae.

[Frequency of Haemophilus spp. in urinary and and genital tract samples]. / Zastupljenost bakterija roda Haemophilus u uzorcima iz mokracnog i genitalnog sustava.

AIM: To determine the prevalence and antibiotic susceptibility of Haemophilus influenzae and H. parainfluenzae isolated from the urinary and genital tracts. METHODS: Identification of strains bacteria Haemophilus spp. was carried out by using API NH identification system, and antibiotic susceptibility was performed by Kirby-Bauer disk diffusion method. RESULTS: A total number of 50 (0.03%) H. influenzae and 14 (0.01%) H. parainfluenzae (out of 180, 415 samples) were isolated from genitourinary tract. From urine samples of the girls under 15 years of age these bacteria were isolated in 13 (0.88%) and two (0.13%) cases, respectively, and only in one case (0.11%) of the UTI in boys (H. influenzae). In persons of fertile age, it was only H. influenzae bacteria that was found in urine samples of the five women (0.04%) and in three men (0.22%). As a cause of vulvovaginitis, H. influenzae was isolated in four (5.63%), and H. parainfluenzae in two (2.82%) girls. In persons of fertile age, H. influenzae was isolated from 10 (0.49%) smears of the cervix, and in nine (1.74%) male samples. H. parainfluenzae was isolated from seven (1.36%) male samples. (p < 0.01). Susceptibility testing ofH. influenzae and H. parainfluenzae revealed that both pathogens were significantly resistant to cotrimoxasol only (26.0% and 42.9%, respectively). CONCLUSION: In the etiology of genitourinary infections of girls during childhood, genital infections of women in fertile age (especially in pregnant women), and men with cases of epididimytis and/or orchitis, it is important to think about this rare and demanding bacteria in terms of cultivation.