Occurrence, genetic characterization, and antibiotic susceptibility of Cronobacter spp. isolated from low water activity functional foods in Brazil.

Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.

A Single Amino Acid Substitution in Elongation Factor G Can Confer Low-Level Gentamicin Resistance in Neisseria gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.

Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1.

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression.

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea.

Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.

Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.

Gene-Gene Interactions Reduce Aminoglycoside Susceptibility of Pseudomonas aeruginosa through Efflux Pump-Dependent and -Independent Mechanisms.

Pseudomonas aeruginosa causes a wide range of acute and chronic infections. Aminoglycosides are a cornerstone of treatment, but isolates are often resistant. The purpose of this research was to better understand the genetic basis of aminoglycoside resistance in P. aeruginosa. Bioinformatic approaches identified mutations in resistance-associated genes in the clinical isolates of P. aeruginosa. The common mutations were then engineered into the genome of P. aeruginosa reference strain PAO1. Mutations in the elongation factor gene fusA1 caused the biggest reduction in aminoglycoside susceptibility, with mutations in the two-component regulator gene amgS and the efflux pump regulator gene mexZ having less impact. This susceptibility was further reduced by combinations of mutations. Mutations in fusA1, amgS and mexZ all increased the expression of the mexXY efflux pump that is strongly associated with aminoglycoside resistance. Furthermore, the fusA1 amgS mexZ triple mutant had the highest efflux pump gene expression. Engineering fusA1 and amgS mutants lacking this efflux pump showed that fusA1 and amgS also reduce aminoglycoside susceptibility through additional mechanisms. The fusA1 and amgS mutations reduced bacterial growth, showing that these mutations have a fitness cost. Our findings demonstrate the complex interplay between mutations, efflux pump expression and other mechanisms for reducing the susceptibility of P. aeruginosa to aminoglycosides.

Effect of Ferredoxin Receptor FusA on the Virulence Mechanism of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is an aerobic Gram-negative bacterium, which is the pathogen of "Visceral white spot disease" in large yellow croaker. P. plecoglossicida is a temperature-dependent bacterial pathogen in fish, which not only reduces the yield of large yellow croaker but also causes continuous transmission of the disease, seriously endangering the healthy development of fisheries. In this study, a mutant strain of fusA was constructed using homologous recombination technology. The results showed that knockout of P. plecoglossicida fusA significantly affected the ability of growth, adhesion, and biofilm formation. Temperature, pH, H2O2, heavy metals, and the iron-chelating agent were used to treat the wild type of P. plecoglossicida; the results showed that the expression of fusA was significantly reduced at 4°C, 12°C, and 37°C. The expression of fusA was significantly increased at pH 4 and 5. Cu2+ has a significant inducing effect on the expression of fusA, but Pb2+ has no obvious effect; the expression of fusA was significantly upregulated under different concentrations of H2O2. The expression of the fusA gene was significantly upregulated in the 0.5~4-µmol/l iron-chelating agent. The expression level of the fusA gene was significantly upregulated after the logarithmic phase. It was suggested that fusA included in the TBDR family not only was involved in the transport of ferredoxin but also played important roles in the pathogenicity and environment adaptation of P. plecoglossicida.

A Master Regulator of Bacteroides thetaiotaomicron Gut Colonization Controls Carbohydrate Utilization and an Alternative Protein Synthesis Factor.

Microbial colonization of the mammalian gut is largely ascribed to the ability to utilize nutrients available in that environment. To understand how beneficial microbes establish a relationship with their hosts, it is crucial to determine what other abilities promote gut colonization. We now report that colonization of the murine gut by the beneficial microbe Bacteroides thetaiotaomicron requires activation of a putative translation factor by the major transcriptional regulator of gut colonization and carbohydrate utilization. To ascertain how this regulator-called BT4338-promotes gut colonization, we identified BT4338-regulated genes and BT4338-bound DNA sequences. Unexpectedly, the gene whose expression was most reduced upon BT4338 inactivation was fusA2, specifying a putative translation factor. We determined that fusA2 activation by BT4338 is conserved in another Bacteroides species and essential for gut colonization in B. thetaiotaomicron because a mutant lacking the BT4338 binding site in the fusA2 promoter exhibited a colonization defect similar to that of a mutant lacking the fusA2 gene. Furthermore, we demonstrated that BT4338 promotes gut colonization independently of its role in carbohydrate utilization because the fusA2 gene was dispensable for utilization of carbohydrates that depend on BT4338 Our findings suggest that microbial gut colonization requires the use of alternative protein synthesis factors.IMPORTANCE The bacteria occupying the mammalian gut have evolved unique strategies to thrive in their environment. Bacteroides organisms, which often comprise 25 to 50% of the human gut microbiota, derive nutrients from structurally diverse complex polysaccharides, commonly called dietary fibers. This ability requires an expansive genetic repertoire that is coordinately regulated to achieve expression of those genes dedicated to utilizing only those dietary fibers present in the environment. Here we identify the global regulon of a transcriptional regulator necessary for dietary fiber utilization and gut colonization. We demonstrate that this transcription factor regulates hundreds of genes putatively involved in dietary fiber utilization as well as a putative translation factor dispensable for growth on such nutrients but necessary for survival in the gut. These findings suggest that gut bacteria coordinate cellular metabolism with protein synthesis via specialized translation factors to promote survival in the mammalian gut.

Efficient differentiation of Nocardia farcinica, Nocardia cyriacigeorgica and Nocardia beijingensis by high-resolution melting analysis using a novel locus.

Accurate identification of Nocardia species remains a challenge due to the complexities of taxonomy and insufficient discriminatory power of traditional techniques. We report the development of a molecular technique that utilizes real-time PCR-based high-resolution melting (HRM) analysis for differentiation of the most common Nocardia species. Based on a novel fusA-tuf intergenic region sequence, Nocardia farcinica, Nocardia cyriacigeorgica and Nocardia beijingensis were clearly distinguished from one another by HRM analysis. The limit of detection of the HRM assay for purified Nocardia spp. DNA was at least 10 fg. No false positives were observed for specificity testing of 20 non-target clinical samples. In comparison to established matrix-assisted laser desorption/ionization-time of flight MS, the HRM assay improved the identification of N. beijingensis. Additionally, all the products of PCR were verified by direct sequencing. In conclusion, the developed molecular assay allows simultaneous detection and differentiation of N. farcinica, N. cyriacigeorgica and N. beijingensis with high sensitivity and specificity.

Isolation, molecular and phenotypic characterization of Cronobacter spp. in ready-to-eat salads and foods from Japanese cuisine commercialized in Brazil.

The aim of this study was to detect Cronobacter from 30 samples of ready-to-eat (RTE) salads and 30 foods from Japanese cuisine as commercially available in Brazil. The detection of Cronobacter was as according to the ISO standard 22964:2017. The isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile was determined using the standardized agar disc diffusion method. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, multiplex-PCR targeting cgcA gene, and fusA allele sequencing. Twenty-seven samples (45.0%) contained Cronobacter, 14 (23.3%) samples of foods from Japanese cuisine and 13 (21.7%) samples of RTE salads. Twenty-nine unique Cronobacter isolates were selected from the 27 positive samples and were identified as C. sakazakii (n = 18), C. malonaticus (n = 8), and C. dublinensis (n = 3). A high genetic diversity was observed, with 29 Cronobacter strains being assigned to 11 different fusA alleles, a ratio of 2.6 strains by fusA allele was found. The cgcA multiplex-PCR failed to identify many of the Cronobacter isolates at the species level. Four (13.8%) Cronobacter isolates were resistant to one or more antibiotics tested (n = 12). The presence of Cronobacter in RTE foods could be a potential threat to human health and highlights the need for high levels of hygiene, particularly when preparing food for elderly, immunosuppressed persons or adults with prior underlying pathology. Epidemiological surveillance agencies should be aware of the risk that these RTE foods may represent, for these groups.

Two new species of Lobellini from Tianmu Mountain, China (Collembola, Neanuridae).

Three species of the subfamily Neanurinae (Collembola: Neanuridae) are recorded from Tianmu Mountain, Zhejiang Province, east China. Two of them, Lobellina fusasp. n. and Paralobella tianmunasp. n., are new to science and described in this paper. Lobellina fusasp. n. can be recognized by the presence of six teeth on mandible and the fusion of dorsointernal tubercles on the head. Paralobella tianmunasp. n. is characterized by a mandible with seven teeth, the lateral tubercle of Abd. II-III respectively with 7 (6+s) chaetae. Crossodonthina bidentata Luo & Chen, 2009 is widely distributed in the mountain from 300 to 1500 m a.s.l.

The cost-effectiveness of 5-FU-SA in the treatment of actinic keratoses of the face and scalp in the UK secondary care setting.

OBJECTIVE: The objective of this analysis was to estimate the relative cost-effectiveness of Actikerall 1 (5-FU-SA) vs cryotherapy in a secondary care setting in the UK, for lesion-directed treatment in patients with actinic keratoses (AK) of the face and scalp. METHODS: The model was a simple decision tree, with a 6-month time horizon. The perspective was that of the UK National Health Service (NHS). Modeled treatment effects included reported per-patient histological clearance and recurrence rates. Cost inputs comprised professional consultation time and cost of medication. Health-related utility estimation followed previously published methodology. Adverse events were not modeled. The key data and model structural assumptions followed expected UK practice. One-way and probabilistic sensitivity analyses were conducted to assess structural and parameter uncertainty. RESULTS: 5-FU-SA was found to be less costly (-£204) and more effective (+0.001 QALY) in base case and sensitivity analyses. In the probabilistic analysis there was 100% probability of being cost-effective over cryotherapy at £20,000 willingness to pay. Cost of professional time was a key driver of the model outcome. 5-FU-SA remained dominant across a range of scenario analyses, including exploration of assumptions around setting of care. LIMITATIONS: The time horizon of the analysis was short and data were not extrapolated beyond the duration of the clinical trial; however, this approach is consistent with likely follow-up of an AK patient. The clinical outcomes observed in the trial were based on a large proportion of cryotherapy patients undergoing an additional cycle of treatment; this may not occur or be required in an experienced secondary care setting. CONCLUSION: 5-FU-SA could be considered as a cost-effective choice for treatment of AK lesions of the face and scalp in secondary and mixed care settings in the UK. Use of 5-FU-SA in patients who would otherwise be managed with cryotherapy has the potential to result in cost savings.

Failure of combination therapy for Staphylococcus aureus bone infection: a case of in vivo selection with resistance to rifampicin and fusidic acid.

Staphylococcus aureus is one of the main etiologies of bone and device-related infections. Treatment of these orthopedic infections combines mostly rifampicin with other antibiotics. The recurrence or failure rate after fusidic acid/rifampicin treatment remains low (<10%). We discuss here a case of antibiotic treatment failure for Staphylococcus aureus bone infection with in vivo selection of rifampicin and fusidic acid resistance. We also report a new mutation in fusA gene involved in fusidic acid resistance.

Síndrome de forestier: caso clínico y revisión de la literatura / Forestier syndrome: clinical case and literature review

Antecedentes: El síndrome de Forestier también conocido como Hiperostosis Esquelética Idiopática Difusa (DISH, por sus siglas en inglés), es una enfermedad de etiología desco-nocida que se caracteriza por osificación del ligamento espinal anterior, siendo las porciones cervicales y torácicas las que se afectan más frecuentemente. Esta enfermedad es más frecuente en hombres y se asocia con diabetes, hipertensión arterial, disli-pidemia y trastornos endocrinos. Descripción del caso clínico:Paciente femenina de 63 años con antecedente de dolor cervical desde hace 32 años, que 6 años después del inicio del cuadro, presentó limitación en la movilidad del cuello; presentando va-rios episodios de disfonía desde hace 10 años; al momento de la consulta la paciente presentó limitación de la movilidad del cue-llo y dolor cervical. La imagen de resonancia magnética reportó: presencia de crecimiento óseo anterior de los cuerpos vertebra-les, este hallazgo está en relación con el síndrome de Forestier. Conclusiones: Por ser una enfermedad poco conocida es sub-diagnosticada y a menudo confundida con otras patologías. Los pacientes son diagnosticados muchos años después de que apa-recieron los primeros síntomas que incluyen dolor, limitación de la movilidad, disfagia y dificultad respiratoria. El tratamiento incluye manejo sintomático, terapia física y manejo quirúrgico...(AU)

Micosis superficiales diagnosticadas en el Laboratorio de micología médica de la Universidad de Costa Rica / Superficial mycosis diagnosed in the laboratory of medical mycology of Costa Rica University

Las micosis superficiales son causa frecuente de consulta, tanto en los servicios de dermatología como en medicina general. En estas afecciones resulta de gran interés el realizar los estudios microbiológicos para hacer el diagnóstico diferencial y para conocer el agente etiológico causante de la patología, no sólo por los aspectos epidemiológicos que esto implica, sino también por el tratamiento. En este estudio se recolectaron 265 muestras de piel y uñas de personas que acudieron al servicio de diagnóstico de la Sección de Micología Médica de la Facultad de Microbiología, Universidad de Costa Rica. Las afecciones en uñas representaron el 67,5 por ciento de los casos atendidos. Trichophyton rubrum es el hongo más aislado; otros dermatofitos y Candida sp también se aislaron de uñas y de piel. Fusarium sp, que es un hongo filamentoso no dermatofito, se aisló de uñas de manos y pies. Este hallazgo es relevante, dado que Fusarium sp., como agente emergente de onicomicosis, no responde bien al tratamiento con fluconazol.

Superficial mycosis is a frequent cause of consultation in Dermatology and Gene- ral Medicine. The microbiological stu- dies of this fungal condition are important not only to do the differential diagnosis, but also to establish the causing agent of the disease as well as its epidemiological aspects and the treatment. In this study 265 skin and nail samples have been collected from individuals at the Laboratory of Medical Mycology of School of Microbiology, Costa Rica University. Nail diseases represent 67,5% of the total cases studied. Trichophyton rubrum was the most frequent isolated fungus; also, other dermatophytes and Candida sp. were isolated from nail and skin. Fusarium sp., a filamentous non-dermatophyte fungus, was isolated from both finger and toe nails. This fin- ding is of relevant, since Fusarium sp., an emergent etiological agent of onychomycosis, does not respond well to treatment based on fluconazol.