Defining standard and high dosages for ß-lactam agents administered by intermittent, prolonged or continuous infusion: a PK/PD simulation study.

BACKGROUND: The new definitions of antimicrobial susceptibility categories proposed by EUCAST in 2020 require the definition of standard and high dosages of antibiotic. For injectable ß-lactams, standard and high dosages have been proposed for short-infusion regimens only. OBJECTIVES: To evaluate dosages for ß-lactams administered by prolonged infusion (PI) and continuous infusion (CI). METHODS: Monte Carlo simulations were performed for seven injectable ß-lactams: aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, piperacillin and temocillin. Various dosage regimens based on short infusion, PI or CI were simulated in virtual patients. Pharmacokinetic (PK) profiles and PTAs were obtained based on reference population PK models, as well as PK/pharmacodynamic targets and MIC breakpoints proposed by EUCAST. Alternative dosage regimens associated with PTA values similar to those of recommended dosages up to the breakpoints were considered acceptable. RESULTS: Adequate PTAs were confirmed for most EUCAST short-infusion dosage regimens. A total of 9 standard and 14 high dosages based on PI (3 to 4 h) or CI were identified as alternatives. For cefepime and aztreonam, only PI and CI regimens could achieve acceptable PTAs for infections caused by Pseudomonas spp.: 2 g q8h as PI of 4 h or 6 g/24 h CI for cefepime; 2 g q6h as PI of 3 h or 6 g/24 h CI for aztreonam. CONCLUSIONS: These alternative standard and high dosage regimens are expected to provide antibiotic exposure compatible with new EUCAST definitions of susceptibility categories and associated MIC breakpoints. However, further clinical evaluation is necessary.

HU Knew? Bacillus subtilis HBsu Is Required for DNA Replication Initiation.

The prokaryotic nucleoid-associated protein (NAP) HU is both highly conserved and ubiquitous. Deletion of HU causes pleiotropic phenotypes, making it difficult to uncover the critical functions of HU within a bacterial cell. In their recent work, Karaboja and Wang (J Bacteriol 204:e00119-22, 2022, https://doi.org/10.1128/JB.00119-22) show that one essential function of Bacillus subtilis HU (HBsu) is to drive the DnaA-dependent initiation of DNA replication at the chromosome origin. We discuss the possible roles of HBsu in replication initiation and other essential cellular functions.

Volcanism and palaeoclimate change drive diversification of the world's largest whip spider (Amblypygi).

The tropics contain many of the most biodiverse regions on Earth but the processes responsible for generating this diversity remain poorly understood. This study investigated the drivers of diversification in arthropods with stenotopic ecological requirements and limited dispersal capability using as a model the monotypic whip spider (Amblypygi) genus Acanthophrynus, widespread in the tropical deciduous forests of Mexico. We hypothesized that for these organisms, the tropical deciduous forests serve as a conduit for dispersal, with their disappearance imposing barriers. Given that these forests are located in a region of complex geological history and that they fluctuated in extent during the Pliocene-Pleistocene glacial/interglacial cycles we combine molecular divergence dating, palaeoclimatic niche modelling and ancestral area reconstruction to test if and when habitat fragmentation promoted diversification in Acanthophrynus. Concomitant with the expected role of landscape change, we demonstrate that orogeny of the Trans-Mexican Volcanic Belt, in the Late Miocene or Early Pliocene (6.95-5.21 million years ago), drove the earliest divergence of Acanthophrynus by vicariance. Similarly, as expected, the later onset of glaciations strongly impacted diversification. Whereas a more stable climate in the southern part of the distribution enabled further diversification, a marked loss of suitable habitat during the glaciations only allowed dispersal and diversification in the north to occur later, resulting in a lower overall diversity in this region. Barriers and diversification patterns identified in Acanthophrynus are reflected in the phylogeography of codistributed vertebrates and arthropods, emphasizing the profound impact of Trans-Mexican Volcanic Belt orogeny and glacial/interglacial cycles as drivers of diversification in the Mexican Neotropics.

Revised version (INFD-D-20-00242): impact of 16S rDNA sequencing on clinical treatment decisions: a single center retrospective study.

BACKGROUND: PCRs targeting 16S ribosomal DNA (16S PCR) followed by Sanger's sequencing can identify bacteria from normally sterile sites and complement standard analyzes, but they are expensive. We conducted a retrospective study in the Strasbourg University Hospital to assess the clinical impact of 16S PCR sequencing on patients' treatments according to different sample types. METHODS: From 2014 to 2018, 806 16S PCR samples were processed, and 191 of those were positive. RESULTS: Overall, the test impacted the treatment of 62 of the 191 patients (32%). The antibiotic treatment was rationalized in 31 patients (50%) and extended in 24 patients (39%), and an invasive procedure was chosen for 7 patients (11%) due to the 16S PCR sequencing results. Positive 16S PCR sequencing results on cerebrospinal fluid (CSF) had a greater impact on patients' management than positive ones on cardiac valves (p = 0.044). The clinical impact of positive 16S PCR sequencing results were significantly higher when blood cultures were negative (p < 0.001), and this difference appeared larger when both blood and sample cultures were negative (p < 0.001). The diagnostic contribution of 16S PCR was higher in patients with previous antibiotic treatment (p < 0.001). CONCLUSION: In all, 16S PCR analysis has a significant clinical impact on patient management, particularly for suspected CSF infections, for patients with culture-negative samples and for those with previous antibiotic treatments.

Management of patients with pulmonary mycobacteriosis in France: a multicenter retrospective cohort study.

BACKGROUND: Recent studies report very low adherence of practitioners to ATS/IDSA recommendations for the treatment of nontuberculous mycobacteria pulmonary disease (NTM-PD), as well as a great variability of practices. Type of management could impact prognosis. METHODS: To evaluate management and prognosis of patients with NTM-PD cases with respect to ATS recommendations, we conducted a multicenter retrospective cohort study (18 sentinel sites distributed throughout France), over a period of six years. We collected clinical, radiological, microbiological characteristics, management and outcome of the patients (especially death or not). RESULTS: 477 patients with NTM-PD were included. Respiratory comorbidities were found in 68% of cases, tuberculosis sequelae in 31.4% of patients, and immunosuppression in 16.8% of cases. The three most common NTM species were Mycobacterium avium complex (60%), M. xenopi (20%) and M. kansasii (5.7%). Smear-positive was found in one third of NTM-PD. Nodulobronchiectatic forms were observed in 54.3% of cases, and cavitary forms in 19.1% of patients. Sixty-three percent of patients were treated, 72.4% of patients with smear-positive samples, and 57.5% of patients with smear-negative samples. Treatment was in adequacy with ATS guidelines in 73.5%. The 2-year mortality was 14.4%. In the Cox regression, treatment (HR = 0.51), age (HR = 1.02), and M. abscessus (3.19) appeared as the 3 significant independent prognostic factors. CONCLUSION: These findings highlight the adequacy between French practices and the ATS/IDSA guidelines. Treatment was associated with a better survival.

Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus.

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole-inheriting daughter cells, resulting in aggregation-free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time-lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short-lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long-lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.

An essential regulatory function of the DnaK chaperone dictates the decision between proliferation and maintenance in Caulobacter crescentus.

Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the α-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor σ32, demonstrating that the DnaK-dependent inactivation of σ32 is a growth requirement. While most mutations occurred in the rpoH gene encoding σ32, we also identified mutations affecting σ32 activity or stability in trans, providing important new insight into the regulatory mechanisms controlling σ32 activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of σ32, and a mutation leading to increased levels of the house keeping σ70 that outcompete σ32 for binding to the RNA polymerase. We demonstrate that σ32 inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has co-opted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.

Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

Pathogenic features of clinically significant coagulase-negative staphylococci in hospital and community infections in Benin.

In West Africa, very little consideration has been given to coagulase negative Staphylococci (CNS). Herein, we describe the features contributing to the pathogenicity of 99 clinically-significant independent CNS isolates associated with infections encountered at the National Teaching Hospital Center of Cotonou (Benin). The pathogenic potentials of nosocomial strains were compared with community strains. S. haemolyticus (44%), S. epidermidis (22%) and S. hominis (7%) were the most frequently isolated while bacteremia (66.7%) and urinary tract infections (24.2%) were the most commonly encountered infections. Most strains were resistant to multiple antibiotics, including penicillin (92%), fosfomycin (81%), methicillin (74%) and trimethoprim-sulfamethoxazole (72%). The most frequently isolated species were also the most frequently resistant to methicillin: S. hominis (100%), S. haemolyticus (93%) and S. epidermidis (67%). Screening of toxic functions or toxin presence revealed hemolytic potential in 25% of strains in over 50% of human erythrocytes in 1h. Twenty-six percent of strains exhibited protease activity with low (5%), moderate (10%) and high activity (11%), while 25% of strains displayed esterase activity. Three percent of strain supernatants were able to lyse 100% of human polymorphonuclear cells after 30min. Polymerase chain reaction and latex agglutination methods revealed staphylococcal enterotoxin C gene expression in 9% of S. epidermidis. A majority of hospital-associated CNS strains (68%) had at least one important virulence feature, compared with only 32% for community-acquired strains. The present investigation confirms that these microorganisms can be virulent, at least in some individual cases, possibly through genetic transfer from S. aureus.

Implementation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis.

The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Staphylococcus aureus and 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 as S. aureus and 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized Staphylococcus species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis strains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified species S. pettenkoferi and putative virulent species, including S. lugdunensis.

Clinical features and treatment outcomes of bone and joint nontuberculous mycobacterial infections according to immune status: a 9-year retrospective observational cohort.

OBJECTIVES: Nontuberculous mycobacteria (NTM) bone and joint infections (BJIs) are uncommon. We evaluated the characteristics of BJIs and identified differences according to immune status. METHODS: We performed a multicenter retrospective study in France involving patients with documented NTM BJI over a 9-year period. We collected the clinical and microbiological characteristics, management, and clinical outcomes of the patients. RESULTS: Overall, 95 patients were included, of whom 50.5% (48/95) were immunosuppressed. Tenosynovitis was more frequent in the immunocompetent group, and native arthritis more common in the immunosuppressed group. Mycobacerium marinum and M. abscessus complex were significantly more frequent in the immunocompetent group, and M. avium and M. xenopi were significantly more frequent in the immunosuppressed group. The combination of antibiotherapy with surgery tended to be more frequent in the immunocompetent than the immunosuppressed group (63.8% (30/47) vs 47.8% (22/46), respectively); of the latter, 45.7% (21/46) received antimicrobial therapy alone, a higher frequency than in the immunocompetent group (23.4%, 11/47). The median duration of antimicrobial treatment was similar in the two groups (11 months). Mortality was significantly higher in the immunosuppressed group. CONCLUSIONS: Although the clinical presentations and the NTM species involved in BJI differed according to immune status, most recovered completely after treatment.

[Lyme borreliosis]. / Borréliose de Lyme.

LYME BORRELIOSIS. Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere, caused by the bacterium Borrelia burgdorferi sensu lato, transmitted to humans by a bite of ticks Ixodes. Prevention is based on simple measures to evict ticks, and on their rapid extractionin the event of a bite. The diagnosis of LB is based on 3 arguments: an exposure to tick bites; clinically compatible symptoms (cutaneous, neurological or rheumatological manifestations, +/- functional symptoms such as fatigue or polyarthromyalgia), evolving in 3 stages (early localized or erythema migrans, early or late disseminated LB); a positive two-tier serological test (ELISA +/- Western-Blot). Serology can be negative for the first 6 weeks, without excluding the diagnosis. Since serology can remain positive for life, evolution is only evaluated clinically. LB treatment is mainly based on doxycycline for 14 to 28 days, depending on the clinical stage and manifestations, without demonstrated interest in prolonging it, even if symptoms persist. Nonetheless their management is crucial as often responsible for medical wandering. Attentive listening to the patient is essential. The prognosis of LB in the medium-term is favorable, especially if they beneficiate of an early management.

BORRÉLIOSE DE LYME. La borréliose de Lyme (BL) est la maladie vectorielle la plus fréquente de l'hémisphère Nord. Elle est due à la bactérie Borrelia burgdorferi sensu lato, transmise à l'homme lors d'une piqûre de tique infectée du genre Ixodes. La prévention repose sur des mesures simples d'éviction des tiques, et sur leur extraction rapide en cas de piqûre. Le diagnostic de la BL est basé sur un trépied : une exposition aux piqûres de tiques ; une clinique compatible (manifestations cutanées, neurologiques ou articulaires, éventuellement accompagnées de symptômes fonctionnels comme une fatigue, des polyarthromyalgies…), évoluant en trois phases (localisée précoce ou érythème migrant, disséminée précoce et tardive) ; une sérologie positive en deux temps (ELISA +/- western-blot). La sérologie peut être négative les 6 premières semaines, sans exclure le diagnostic. La sérologie pouvant rester positive à vie, l'évolution est uniquement évaluée cliniquement. Le traitement de la BL repose principalement sur la doxycycline, pendant 14 à 28 jours selon le stade clinique et le type d'atteinte. Il n'y a pas d'intérêt démontré à prolonger l'antibiothérapie, même en cas de persistance de symptômes. Néanmoins la prise en charge de ceux-ci (réadaptation physique, thérapies brèves, etc.) est fondamentale car ils sont souvent à l'origine d'une errance médicale. Une écoute attentive du patient est essentielle. Le pronostic des BL à moyen terme est favorable, ce d'autant que leur prise en charge est précoce.

The bacterial replication origin BUS promotes nucleobase capture.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

Molecular detection of isoniazid monoresistance improves tuberculosis treatment: A retrospective cohort in France.

OBJECTIVES: Isoniazid-monoresistant tuberculosis (HR-TB) requires early diagnosis and adapted treatment to achieve optimal outcomes. The primary aim of the study was to assess the impact of the implementation of rapid diagnostic tests on HR-TB treatment in France. METHODS: We designed a retrospective multicentre study including consecutive HR-TB patients diagnosed in 2016 and 2017. Implementation of a molecular assay detecting isoniazid resistance directly on a clinical sample was recorded. The association between early implementation of such assays and adequate treatment was assessed by a multivariable Cox proportional hazards model. RESULTS: Overall, 99 HR-TB patients were included from 20 University Hospitals. Among all smear-positive HR-TB patients, only 26% beneficiated from early molecular HR detection. This detection was independently associated with shorter time to adequate treatment (HR = 2.0 [1.1-3.8], p = 0.03). CONCLUSION: In our study, molecular detection of HR on an initial sample was independently associated with earlier treatment adaptation.

Antialarmin effect of tick saliva during the transmission of Lyme disease.

Tick saliva has potent immunomodulatory properties. In arthropod-borne diseases, this effect is largely used by microorganisms to increase their pathogenicity and to evade host immune responses. We show that in Lyme borreliosis, tick salivary gland extract and a tick saliva protein, Salp15, inhibit in vitro keratinocyte inflammation induced by Borrelia burgdorferi sensu stricto or by the major outer surface lipoprotein of Borrelia, OspC. Chemokines (interleukin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]) and several antimicrobial peptides (defensins, cathelicidin, psoriasin, and RNase 7) were downregulated. Interestingly, antimicrobial peptides (AMPs) transiently inhibited bacterial motility but did not kill the organisms when tested in vitro. We conclude that tick saliva affects the chemotactic properties of chemokines and AMPs on immune cells and has an antialarmin effect on human primary keratinocytes. Alarmins are mediators that mobilize and activate antigen-presenting cells. Inhibition of cutaneous innate immunity and of the migration of immune cells to the site of the tick bite ensures a favorable environment for Borrelia. The bacterium can then multiply locally and, subsequently, disseminate to the target organs, including joints, heart, and the central nervous system.