Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

Ruptured Appendicitis and Retrocecal Abscess Masquerading as Knee Pain in a Pediatric Patient: A Case Report.

BACKGROUND: Knee pain has a variety of etiologies in the pediatric population, including septic arthritis, osteomyelitis, fracture, ligamentous injury, and neoplasms. Extrinsic sources of knee pain may also be intra-abdominal, although abdominal pathology is much more likely to manifest as hip or proximal thigh musculature pain. CASE REPORT: A 5-year-old healthy male presented with atraumatic right knee pain, discomfort with weightbearing, fever, and elevated inflammatory laboratory markers. Physical examination and magnetic resonance imaging findings of the knee were benign, leading to low clinical suspicion for knee septic arthritis. Blood cultures were positive for a gastrointestinal organism, Granulicatella adiacens, suggesting abdominal pathology leading to referred pain. Ultrasound evaluation and computed tomography (CT) of the abdomen revealed a large abscess secondary to perforated appendicitis, which was treated with CT-guided drainage and i.v. antibiotics. The patient's musculoskeletal pain subsided with treatment of the appendicitis. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Acute appendicitis may present as knee pain, with other signs and symptoms mimicking septic arthritis, such as fever, inability to bear weight, and elevated inflammatory markers. Considering an array of differential diagnoses in pediatric patients with apparent knee septic arthritis is crucial to prevent delay in diagnosis of alternative infectious sources.

In vitro antimicrobial activity against Abiotrophia defectiva and Granulicatella elegans biofilms.

OBJECTIVES: To determine the efficacy of different antibiotics (alone or in combination) against Abiotrophia defectiva and Granulicatella elegans biofilms and to investigate the anti-biofilm activity of gentamicin alone versus blood culture isolates from both species. METHODS: The activity of benzylpenicillin, clindamycin, daptomycin, fosfomycin, gentamicin, levofloxacin and rifampicin against 24-hour-old biofilms of A. defectiva and G. elegans was investigated in vitro by conventional microbiological methods and isothermal microcalorimetry. RESULTS: For planktonic bacteria, the MIC values of tested antibiotics ranged from 0.016 to 64 mg/L, as determined by microcalorimetry. Higher antibiotic concentrations, ranging from 1 to >1024 mg/L, were needed to produce an effect on biofilm bacteria. Gentamicin was an exception as it was active at 1 mg/L against both planktonic and biofilm G. elegans. A synergistic effect was observed when daptomycin was combined with benzylpenicillin, gentamicin or rifampicin against A. defectiva biofilms and when gentamicin was combined with rifampicin or levofloxacin against G. elegans biofilms. A. defectiva clinical isolates displayed greater variability in gentamicin susceptibility as compared with G. elegans strains. CONCLUSIONS: Antimicrobial susceptibility profiles vary widely between Abiotrophia and Granulicatella biofilms, and synergistic effects of the tested antibiotics were heterogeneous. The clinical relevance of these in vitro observations needs to be confirmed in experimental in vivo conditions and human trials, before guidelines for the treatment of A. defectiva and G. elegans infections are established. This study suggests the benefit of further clinical exploration of antibiotic combinations with anti-biofilm effect.

Quantitative assessment of individual populations within polymicrobial biofilms.

Selecting appropriate tools providing reliable quantitative measures of individual populations in biofilms is critical as we now recognize their true polymicrobial and heterogeneous nature. Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence in situ hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of Pseudomonas aeruginosa, Inquilinus limosus and Dolosigranulum pigrum was assessed in dual- and triple-species consortia under oxygen and antibiotic stress. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and/or PNA-FISH (≤4 log10 cells/cm2) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg D. pigrum limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method's reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities´ changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections.

Successful treatment of a penicillin-intermediate and ceftriaxone-resistant Granulicatella adiacens presumed prosthetic valve endocarditis with vancomycin.

Advancements in rapid diagnostics have helped to identify nutritionally variant streptococci (NVS) as an increasing cause of infective endocarditis (IE). This case report highlights the challenges in susceptibility testing and the importance of appropriate empiric treatment for Granulicatella adiacens, and provides considerations for future practice guideline recommendations. Guidelines for treatment of IE caused by NVS are currently limited to patients with native valve disease. We present a patient with presumed prosthetic valve endocarditis caused by G. adiacens, with clinically relevant resistance to recommended first-line agents (penicillin and ceftriaxone), who was successfully treated with 8 weeks of intravenous (IV) vancomycin. Vancomycin is currently recommended as an alternate therapy for patients intolerant of penicillins, but we believe vancomycin should be considered a first-line empiric treatment option for IE when the identified organism is G. adiacens and susceptibility testing is not readily available.

Developing a model for cystic fibrosis sociomicrobiology based on antibiotic and environmental stress.

Cystic fibrosis (CF) infections are invariably biofilm-mediated and polymicrobial, being safe to assume that a myriad of factors affects the sociomicrobiology within the CF infection site and modulate the CF community dynamics, by shaping their social activities, overall functions, virulence, ultimately affecting disease outcome. This work aimed to assess changes in the dynamics (particularly on the microbial composition) of dual-/three-species biofilms involving CF-classical (Pseudomonas aeruginosa) and unusual species (Inquilinus limosus and Dolosigranulum pigrum), according to variable oxygen conditions and antibiotic exposure. Low fluctuations in biofilm compositions were observed across distinct oxygen environments, with dual-species biofilms exhibiting similar relative proportions and P. aeruginosa and/or D. pigrum populations dominating three-species consortia. Once exposed to antibiotics, biofilms displayed high resistance profiles, and microbial compositions, distributions, and microbial interactions significantly challenged. The antibiotic/oxygen environment supported such fluctuations, which enhanced for three-species communities. In conclusion, antibiotic therapy hugely disturbed CF communities' dynamics, inducing significant compositional changes on multispecies consortia. Clearly, multiple perturbations may disturb this dynamic, giving rise to various microbiological scenarios in vivo, and affecting disease phenotype. Therefore, an appreciation of the ecological/evolutionary nature within CF communities will be useful for the optimal use of current therapies and for newer breakthroughs on CF antibiotherapy.

Alloiococcus otitidis Forms Multispecies Biofilm with Haemophilus influenzae: Effects on Antibiotic Susceptibility and Growth in Adverse Conditions.

Otitis media with effusion (OME) is a biofilm driven disease and commonly accepted otopathogens, such as Haemophilus influenzae, Streptococcus pneumonia, and Moraxella catarrhalis, have been demonstrated to form polymicrobial biofilms within the middle ear cleft. However, Alloiococcus otitidis (A. otitidis), which is one of the most commonly found bacteria within middle ear aspirates of children with OME, has not been described to form biofilms. The aim of this study was to investigate whether A. otitidis can form biofilms and investigate the impact on antibiotic susceptibility and survivability in polymicrobial biofilms with H. influenzae in vitro. The ability of A. otitidis to form single-species and polymicrobial biofilms with H. influenzae was explored. Clinical and commercial strains of A. otitidis and H. influenzae were incubated in brain heart infusion with and without supplementation. Biofilm was imaged using confocal laser scanning microscopy and scanning electron microscopy. Quantification of biofilm biomass and viable bacterial number was assessed using crystal violet assays and viable cell counting in both optimal growth conditions and in adverse growth conditions (depleted media and sub-optimal growth temperature). Antimicrobial susceptibility and changes in antibiotic resistance of single-species and multi-species co-culture were assessed using a microdilution method to assess minimal bactericidal concentration and E-test for amoxicillin and ciprofloxacin. A. otitidis formed single-species and polymicrobial biofilms with H. influenzae. Additionally, whilst strain dependent, combinations of polymicrobial biofilms decreased antimicrobial susceptibility, albeit a small magnitude, in both planktonic and polymicrobial biofilms. Moreover, A. otitidis promoted H. influenzae survival by increasing biofilm production in depleted media and at suboptimal growth temperature. Our findings suggest that A. otitidis may play an indirect pathogenic role in otitis media by altering H. influenzae antibiotic susceptibility and enhancing growth under adverse conditions.

Antimicrobial Susceptibility Patterns among a Large, Nationwide Cohort of Abiotrophia and Granulicatella Clinical Isolates.

Antimicrobial susceptibility patterns from 599 A. defectiva, G. adiacens, and G. elegans clinical isolates were determined by broth microdilution. We observed significant differences in susceptibility across species, particularly to penicillin and ceftriaxone, and across geographical regions. A. defectiva was the least susceptible species overall to penicillin. All isolates were susceptible to vancomycin and >90% were susceptible to levofloxacin.

Performance of Etest for Antimicrobial Susceptibility Testing of Abiotrophia defectiva and Granulicatella Species.

The Etest on chocolate Mueller-Hinton agar was compared to broth microdilution (BMD) for 125 isolates of nutritionally variant streptococci. Vancomycin Etests yielded 31.1% essential agreement (EA) and 20.0% categorical agreement (CA). Penicillin Etests yielded 86.0% EA and 85.6% CA, whereas ceftriaxone Etests yielded 73.6% EA and 68.0% CA.

Differential Antimicrobial Susceptibilities of Granulicatella adiacens and Abiotrophia defectiva.

MICs of 25 Abiotrophia defectiva and 109 Granulicatella adiacens isolates were determined by broth microdilution. Using CLSI breakpoints, the susceptibilities of A. defectiva and G. adiacens isolates were, respectively, 24% and 34% to penicillin, 92% and 22% to ceftriaxone, 48% and 3% to cefepime, 72% and 87% to meropenem, 92% and 10% to cefotaxime, 100% and 97% to levofloxacin, 92% and 80% to clindamycin, and 24% and 50% to erythromycin. All isolates were susceptible to vancomycin. In the penicillin-susceptible subgroup, all A. defectiva isolates were susceptible to ceftriaxone; however, 62% of G. adiacens isolates were ceftriaxone nonsusceptible.

Antimicrobial Susceptibilities of Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans.

Nutritionally variant streptococci (NVS) are fastidious Gram-positive cocci comprised of the species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans. NVS are an important cause of bacteremia and infective endocarditis (IE) associated with significant morbidity and mortality. Antimicrobial susceptibility testing (AST) was performed for 14 antimicrobials using the broth microdilution MIC method described in the Clinical and Laboratory Standards Institute (CLSI) M45 guideline. A total of 132 clinical NVS blood isolates collected from 2008 to 2014 were tested. Species level identification of NVS isolates was achieved by 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Ninety isolates were identified as G. adiacens, 37 as A. defectiva, and 5 as G. elegans. All isolates were susceptible to vancomycin (MIC90 = 1 µg/ml), and none displayed high-level resistance to aminoglycosides. G. adiacens was considerably more susceptible to penicillin than A. defectiva (38.9% versus 10.8% of isolates susceptible) but was less susceptible to cephalosporins than was A. defectiva (43.3% versus 100% of isolates susceptible to ceftriaxone). Several isolates were resistant to levofloxacin (6%), erythromycin (51%), and clindamycin (10%). The MIC90 for daptomycin was ≥ 4 µg/ml for G. adiacens and A. defectiva. G. elegans isolates were 100% susceptible to all antimicrobials tested, with the exception of erythromycin, to which only 20% were susceptible. This study provides antimicrobial susceptibility data for a recent collection of NVS and demonstrates important NVS species-related differences with respect to susceptibility to penicillin, cephalosporins, carbapenems, and daptomycin. Species-level identification of NVS organisms when susceptibility testing is not readily available may aid in treatment decisions.

In vitro inflammatory responses elicited by isolates of Alloiococcus otitidis obtained from children with otitis media with effusion.

Alloiococcus otitidis is usually detected in children with otitis media (OM) by PCR as it is not often detected by routine culture. Our improved method for its isolation obtained A. otitidis from nearly 50% of 78 children with OM with effusion. The role of A. otitidis in pathogenesis of OM is unclear. This study tested two hypothesis: (1) that fresh isolates of A. otitidis would elicit pro-inflammatory cytokines from THP-1 monocytic cells equivalent to those induced by Streptococcus pneumoniae; (2) priming THP-1 cells with interferon-gamma (IFN-γ) a surrogate for virus infection, would enhance pro-inflammatory responses. Recent clinical isolates of A. otitidis, S. pneumoniae (ATCC 49619) and a blood culture isolate of S. pneumoniae (SP2) were used in the assays. Cytokines were quantified by BioRad bead assay and Luminex 200. IFN-γ priming enhanced cytokine responses. S. pneumoniae ATCC 49619 induced lower responses than SP2 for IL-1ß, IL-6, TNF-α. A. otitidis LW 27 elicited higher IL-1ß and TNF-α responses than either pneumococcal isolate. Small green colony types of A. otitidis induced higher responses than large white colony types for IL-8 and IL-1ß. The hypothesis that A. otitidis elicits cytokines observed in middle ear effusions was supported; the need to use recent clinical isolates in studies of pathogenesis was highlighted.

Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections.

Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens.

Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues.

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure-activity relationship in the N-terminal domain of these peptides, three analogues (1-3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal ß-sheet residues of the native peptide with shorter ß-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into ß-sheet conformation. These structure-activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.

Antibiotic resistance of mixed biofilms in cystic fibrosis: impact of emerging microorganisms on treatment of infection.

Cystic fibrosis (CF) is a genetic disorder associated with multispecies infections where interactions between classical and newly identified bacteria might be crucial to understanding the persistent colonisation in CF lungs. This study investigated the interactions between two emerging species, Inquilinus limosus and Dolosigranulum pigrum, and the conventional CF pathogen Pseudomonas aeruginosa by evaluating the ability to develop biofilms of mixed populations and then studying their susceptibility patterns to eight different antimicrobials. Monospecies biofilms formed by I. limosus and D. pigrum produced significantly less biomass than P. aeruginosa and displayed greater sensitivity to antimicrobials. However, when in dual-species biofilms with P. aeruginosa, the emerging species I. limosus and D. pigrum were crucial in increasing tolerance of the overall consortia to most antibiotics, even without a change in the number of biofilm-encased cells. These results may suggest that revising these and other species interactions in CF might enable the development of more suitable and effective therapies in the future.