Monkeypox virus genomic accordion strategies.

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

Role of the first WHO mutation catalogue in the diagnosis of antibiotic resistance in Mycobacterium tuberculosis in the Valencia Region, Spain: a retrospective genomic analysis.

BACKGROUND: In June, 2021, WHO published the most complete catalogue to date of resistance-conferring mutations in Mycobacterium tuberculosis. Here, we aimed to assess the performance of genome-based antimicrobial resistance prediction using the catalogue and its potential for improving diagnostics in a real low-burden setting. METHODS: In this retrospective population-based genomic study M tuberculosis isolates were collected from 25 clinical laboratories in the low-burden setting of the Valencia Region, Spain. Culture-positive tuberculosis cases reported by regional public health authorities between Jan 1, 2014, and Dec 31, 2016, were included. The drug resistance profiles of these isolates were predicted by the genomic identification, via whole-genome sequencing (WGS), of the high-confidence resistance-causing variants included in the catalogue and compared with the phenotype. We determined the minimum inhibitory concentration (MIC) of the isolates with discordant resistance profiles using the resazurin microtitre assay. FINDINGS: WGS was performed on 785 M tuberculosis complex culture-positive isolates, and the WGS resistance prediction sensitivities were: 85·4% (95% CI 70·8-94·4) for isoniazid, 73·3% (44·9-92·2) for rifampicin, 50·0% (21·1-78·9) for ethambutol, and 57·1% (34·0-78·2) for pyrazinamide; all specificities were more than 99·6%. Sensitivity values were lower than previously reported, but the overall pan-susceptibility accuracy was 96·4%. Genotypic analysis revealed that four phenotypically susceptible isolates carried mutations (rpoB Leu430Pro and rpoB Ile491Phe for rifampicin and fabG1 Leu203Leu for isoniazid) known to give borderline resistance in standard phenotypic tests. Additionally, we identified three putative resistance-associated mutations (inhA Ser94Ala, katG Leu48Pro, and katG Gly273Arg for isoniazid) in samples with substantially higher MICs than those of susceptible isolates. Combining both genomic and phenotypic data, in accordance with the WHO diagnostic guidelines, we could detect two new multidrug-resistant cases. Additionally, we detected 11 (1·6%) of 706 isolates to be monoresistant to fluoroquinolone, which had been previously undetected. INTERPRETATION: We showed that the WHO catalogue enables the detection of resistant cases missed in phenotypic testing in a low-burden region, thus allowing for better patient-tailored treatment. We also identified mutations not included in the catalogue, relevant at the local level. Evidence from this study, together with future updates of the catalogue, will probably lead in the future to the partial replacement of culture testing with WGS-based drug susceptibility testing in our setting. FUNDING: European Research Council and the Spanish Ministerio de Ciencia.

Clinical outcomes and antibody transfer in a cohort of infants with in utero or perinatal exposure to SARS-CoV-2 (Coronascope Study).

We aimed to describe the outcomes, focusing on the hearing and neurological development, of infants born to mothers with COVID-19 during pregnancy and to evaluate the persistence of maternal antibodies in the first months of life. An observational, prospective study at a tertiary hospital in Madrid (Spain) on infants born to mothers with COVID-19 during pregnancy between March and September 2020 was conducted. A follow-up visit at 1-3 months of age with a physical and neurological examination, cranial ultrasound (cUS), SARS-CoV-2 RT-PCR on nasopharyngeal swab, and SARS-CoV-2 serology were performed. Hearing was evaluated at birth through the automated auditory brainstem response and at six months of age through the auditory steady-state response. A neurodevelopmental examination using the Bayley-III scale was performed at 12 months of age. Of 95 infants studied, neurological examination was normal in all of them at the follow-up visit, as was the cUS in 81/85 (95%) infants, with only mild abnormalities in four of them. Serology was positive in 47/95 (50%) infants, which was not associated with symptoms or severity of maternal infection. No hearing loss was detected, and neurodevelopment was normal in 96% of the infants (median Z score: 0). CONCLUSION: In this cohort, the majority of infants born to mothers with COVID-19 during pregnancy were healthy infants with a normal cUS, no hearing loss, and normal neurodevelopment in the first year of life. Only half of the infants had a positive serological result during the follow-up. WHAT IS KNOWN: • Hearing loss and neurodevelopmental delay in infants born to mothers with COVID-19 during pregnancy has been suggested, although data is inconsistent. Maternal antibody transfer seems to be high, with a rapid decrease during the first weeks of life. WHAT IS NEW: • Most infants born to mothers with COVID-19 during pregnancy had normal hearing screening, cranial ultrasound, and neurodevelopmental status at 12 months of life. Antibodies against SARS-CoV-2 were only detected in 50% of the infants at two months of life.

Pathological Findings Associated With SARS-CoV-2 on Postmortem Core Biopsies: Correlation With Clinical Presentation and Disease Course.

Background: Autopsies can shed light on the pathogenesis of new and emerging diseases. Aim: To describe needle core necropsy findings of the lung, heart, and liver in decedents with COVID-19. Material: Cross-sectional study of needle core necropsies in patients who died with virologically confirmed COVID-19. Histopathological analyses were performed, and clinical data and patient course evaluated. Results: Chest core necropsies were performed in 71 decedents with a median age of 81 years (range 52-97); 47 (65.3%) were men. The median interval from symptoms onset to death was 17.5 days (range 1-84). Samples of lung (n = 62, 87.3%), heart (n = 48, 67.6%) and liver (n = 39, 54.9%) were obtained. Fifty-one lung samples (82.3%) were abnormal: 19 (30.6%) showed proliferative diffuse alveolar damage (DAD), 12 (19.4%) presented exudative DAD, and 10 (16.1%) exhibited proliferative plus exudative DAD. Of the 46 lung samples tested for SARS-CoV-19 by RT-PCR, 39 (84.8%) were positive. DAD was associated with premortem values of lactate dehydrogenase of 400 U/L or higher [adjusted odds ratio (AOR) 21.73; 95% confidence interval (CI) 3.22-146] and treatment with tocilizumab (AOR 6.91; 95% CI 1.14-41.7). Proliferative DAD was associated with an onset-to-death interval of over 15 days (AOR 7.85, 95% CI 1.29-47.80). Twenty-three of the 48 (47.9%) heart samples were abnormal: all showed fiber hypertrophy, while 9 (18.8%) presented fibrosis. Of the liver samples, 29/39 (74.4%) were abnormal, due to steatosis (n = 12, 30.8%), cholestasis (n = 6, 15.4%) and lobular central necrosis (n = 5, 12.8%). Conclusion: Proliferative DAD was the main finding on lung core needle necropsy in people who died from COVID-19; this finding was related to a longer disease course. Changes in the liver and heart were common.

Population-based sequencing of Mycobacterium tuberculosis reveals how current population dynamics are shaped by past epidemics.

Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application. Here, we generate a population-based dataset from Valencia region and compare it with available datasets from different TB-burden settings to reveal transmission dynamics heterogeneity and its public health implications. We sequenced the whole genome of 785 Mycobacterium tuberculosis strains and linked genomes to patient epidemiological data. We use a pairwise distance clustering approach and phylodynamic methods to characterize transmission events over the last 150 years, in different TB-burden regions. Our results underscore significant differences in transmission between low-burden TB settings, i.e., clustering in Valencia region is higher (47.4%) than in Oxfordshire (27%), and similar to a high-burden area as Malawi (49.8%). By modeling times of the transmission links, we observed that settings with high transmission rate are associated with decades of uninterrupted transmission, irrespective of burden. Together, our results reveal that burden and transmission are not necessarily linked due to the role of past epidemics in the ongoing TB incidence, and highlight the need for in-depth characterization of transmission dynamics and specifically tailored TB control strategies.

Nasopharyngeal Microbiota as an early severity biomarker in COVID-19 hospitalised patients.

This study aimed to analyse the diversity and taxonomic composition of the nasopharyngeal microbiota, to determine its association with COVID-19 clinical outcome. To study the microbiota, we utilized 16S rRNA sequencing of 177 samples that came from a retrospective cohort of COVID-19 hospitalized patients. Raw sequences were processed by QIIME2. The associations between microbiota, invasive mechanical ventilation (IMV), and all-cause mortality were analysed by multiple logistic regression, adjusted for age, gender, and comorbidity. The microbiota α diversity indexes were lower in patients with a fatal outcome, whereas the ß diversity analysis showed a significant clustering in these patients. After multivariate adjustment, the presence of Selenomonas spp., Filifactor spp., Actinobacillus spp., or Chroococcidiopsis spp., was associated with a reduction of more than 90% of IMV. Higher diversity and the presence of certain genera in the nasopharyngeal microbiota seem to be early biomarkers of a favourable clinical evolution in hospitalized COVID-19 patients.

A new MALDI-TOF approach for the quick sequence type identification of Legionella pneumophila.

BACKGROUND: Recently, MALDI-TOF has emerged as a quick tool for bacterial typing. The aim was to evaluate if MALDI-TOF based typing of Legionella pneumophila can achieve the same discriminatory power as that of the Sequence Based Typing (SBT) method. METHODS: The Sequence Type (ST) was obtained from the 90 strains included in the training set and an in-house MALDI-TOF library based on the Main Spectra Profile (MSP) was generated for the identification of such ST. Then, our library was validated by three procedures: a) creating a dendrogram, b) searching for specific peaks present exclusively in each MSP entry, and c) analysing a validation set composed of 14 strains with known ST. Fully characterized L. pneumophila ATCC 33152, which belongs to ST 36, was used as a control strain. RESULTS: In the training set, 17 strains belonged to ST 1, 1 to ST 20, 63 to ST 22, 1 to ST 146, 6 to ST 578, and 2 to ST 1086. Specific peaks present in each MSPs spectrum, which are considered type-specific biomarkers, ranged from 2 to 11; more concretely, MSP for ST 1 identification shows 2 specific peaks; MSP for ST 20 identification: 9 specific peaks; MSP for ST 22 and ST 36 identification: 11 specific peaks; MSP for ST 146 identification: 5 specific peaks; and MSP for ST 578 and ST 1086 identification: 3 specific peaks. Using the validation set (nine strains belonging to ST 22 and five to ST 1), MALDI-TOF assigned accurately the ST in 30 min per tested strain with a full match. CONCLUSIONS: The ST of L. pneumophila can be identified and reported in few minutes directly from colonies grown on BCYE agar using MALDI-TOF.

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain.

BACKGROUND: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened. METHODS AND FINDINGS: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region. CONCLUSIONS: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control.

Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013.

Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide.

Legionellosis outbreak associated with asphalt paving machine, Spain, 2009.

From 1999 through 2005 in Alcoi, Spain, incidence of legionellosis was continually high. Over the next 4 years, incidence was lower, but an increase in July 2009 led health authorities to declare an epidemic outbreak. A molecular epidemiology investigation showed that the allelic profiles for all Legionella pneumophila samples from the 2009 outbreak patients were the same, thus pointing to a common genetic origin for their infections, and that they were identical to that of the organism that had caused the previous outbreaks. Spatial-temporal and sequence-based typing analyses indicated a milling machine used in street asphalt repaving and its water tank as the most likely sources. As opposed to other machines used for street cleaning, the responsible milling machine used water from a natural spring. When the operation of this machine was prohibited and cleaning measures were adopted, infections ceased.

Relation between induction of the mar operon and cyclohexane tolerance and reduction in fluoroquinolone susceptibility in Salmonella spp.

The relationship between cyclohexane tolerance and induction of the mar operon and a decrease in susceptibility to ciprofloxacin and moxifloxacin in isolates of Salmonella spp. from food and clinical isolates of Salmonella spp. was studied. We studied the influence of the mar operon using an inductor (acetylsalicylic acid) and we also studied the cyclohexane resistance. Induction was seen to produce an increase in the minimum inhibitory concentration (MIC) of these quinolones, which suggested that there was overexpression of the AcrAB type active efflux systems due to induction of the mar operon. Cyclohexane susceptibility was not shown to be a very sensitive method for studying this process, as only 3% (5/176) of the clinical isolates studied were cyclohexane-resistant; most of these belonged to the Hadar serotype. This study confirmed the participation of active efflux systems in the decrease in fluoroquinolone susceptibility in Salmonella spp. Furthermore, the study has indicated that these mechanisms (i.e., active efflux systems) are present in strains that are susceptible to the fluoroquinolone compounds, so their stimulation may be one of the mechanisms involved in the reduction in fluoroquinolone susceptibility. This suggests that the exposure of Salmonella spp. to antibiotics should be limited in order to prevent these active efflux systems from being activated. Consequently, the use of fluoroquinolones, both in the treatment of humans and in veterinary practice, should be controlled and rationalized in an attempt to curb the increase in the number of strains that are resistant to these compounds.

Effect of exposure to fluoroquinolones and beta-lactams on the in vitro activity of other groups of antibiotics in Salmonella spp.

After designing in vitro models of repeated exposure of Salmonella spp. to various beta-lactams and fluoroquinolones we studied the decrease in susceptibility to other antibiotic families of the mutants generated. There was a decrease in the susceptibility of all the mutants to tetracycline, cotrimoxazole and chloramphenicol. Mutants generated following exposure to fluoroquinolones showed reduced susceptibility to amoxicillin, amoxicillin/clavulanic acid, cefoxitin and cefuroxime, whereas mutants generated following beta-lactam exposure showed reduced susceptibility to nalidixic acid and ciprofloxacin. We observed that the efflux pump systems are activated in the mutants generated and this may therefore be the cause of the decrease in susceptibility. In many cases the decrease is small and is not detected if the CLSI criteria are applied. Nevertheless, more detailed studies should be done to evaluate the importance of this phenomenon and rationalize the use of antibiotics in both humans and animals so as to control the increase in the number of multiresistant strains.

In vitro generation of Salmonella spp. mutants following repeated exposure to beta-lactam antibiotics.

Beta-lactams are a group of drugs considered to be the treatment of choice in cases of salmonellosis, although many isolates that are clinically resistant to these compounds have been reported. We developed an in vitro model to determine the dynamics of the generation of resistant mutants following repeated exposure to these drugs. All the isolates exposed repeatedly to amoxicillin and cefotaxime exhibit a decrease in their susceptibility to these compounds and to many other beta-lactam drugs. However, in many cases this reduction in susceptibility is not detected if the CLSI criteria are applied. These mutants with reduced susceptibility may be the reason for therapeutic failures, so this phenomenon should be studied in greater detail by means of in vitro models or clinical studies.

In vitro activity of linezolid against Staphylococcus aureus: a population study.

In our study, we aim to determine the existence of microorganisms that are heteroresistant to linezolid among Staphylococcus aureus clinical isolates in our setting between 1996 and 2002; during this period, linezolid was not used in clinical practice. There was no resistant subpopulation to 4 mg/l of linezolid in 99.4% of the strains. On the other hand, 16.46% of the strains exhibited resistant subpopulations to 4 microg/ml of vancomycin. However, the emergence of strains resistant to this drug has been described and the emergence of resistant strains should be monitored.

Characterization of Salmonella spp. mutants with reduced fluoroquinolone susceptibility: importance of efflux pump mechanisms.

BACKGROUND: We studied the importance of the efflux pump mechanisms in Salmonella spp. mutants with reduced fluoroquinolone susceptibility generated in vitro. METHODS: The efflux pump was studied using MC-207,110 as an inhibitor of these systems. RESULTS: Wild strains with mutations in gyrA exhibit greater activity of the efflux pump systems than nalidixic-acid-susceptible strains (30-fold). When we evaluate the respective mutants, in those of susceptible strains there is seen to be greater elimination of the antibiotic (13-fold), whereas in mutants of nalidixic-acid-resistant strains these systems are not modified. When evaluating the influence of the antibiotic generating the mutants, ciprofloxacin is seen to be the quinolone that activates the efflux pump systems the most. CONCLUSIONS: Repeated exposure to low concentrations of all the fluoroquinolones studied leads to activation of the efflux pump systems and a reduction in susceptibility, even when there are no mutations in gyrA. Activation of these mechanisms is greatly influenced by the chemical structure of the antibiotic. The capacity of these systems to eliminate fluoroquinolones is limited and therefore, for the microorganism to acquire high-level fluoroquinolone resistance, they must be complemented by other mechanisms.

The importance of active efflux systems in the quinolone resistance of clinical isolates of Salmonella spp.

The aim of this study was to determine the importance of the active elimination of antibiotics by active efflux systems, in the decrease in fluoroquinolone sensitivity of clinical isolates of Salmonella spp. as well as the intrinsic antibiotic activity of certain active efflux system inhibitors. The effect of the active efflux system on the decrease in sensitivity to nalidixic acid, ciprofloxacin, ofloxacin and sparfloxacin was studied by investigating the variation in the in vitro activity of these compounds when assayed in association with reserpine and MC 207.110. The active efflux systems inhibited by reserpine displayed low activity in the elimination of these compounds, whereas those inhibited by MC 207.110 showed high activity in the elimination of nalidixic acid and sparfloxacin, but were less effective in the elimination of ofloxacin and ciprofloxacin. These two compounds did not exhibit intrinsic inhibitory activity against Salmonella spp. at the concentrations assayed. These mechanisms of resistance to antibiotics are complex and vary depending on the chemical composition of the antibiotics used, and perhaps the inhibitors of these systems, although they do not exhibit any intrinsic antibiotic activity, may be used as adjuvants to increase the activity of certain antibiotics. These mechanisms complement the mutations in the gyrA gene and this supports the thesis that it is necessary to lower the breakpoint established by the NCCLS for ciprofloxacin, since the strains studied have resistance mechanisms that reduce the activity of this drug and may favour the emergence of resistant mutants during treatment.

Mutations in the gyrA gene in Salmonella enterica clinical isolates with decreased ciprofloxacin susceptibility.

The incidence of resistance to nalidixic acid has increased in the University General Hospital of Elche, Spain, and this paper describes the investigation of this phenomenon. This increase was mainly due to an increase of nalidixic-resistant Salmonella enterica serotype Enteritidis. Resistance to nalidixic acid is an indicator of decreased ciprofloxacin susceptibility (sensitivity 100%, specificity 96.7%). Strains that were resistant to nalidixic acid and exhibited decreased susceptibility to ciprofloxacin had a single mutation in QRDR of gyrA: Asp87-Asn, Asp87-Tyr or Ser83-Phe. The sensitivity of S. enterica strains to nalidixic acid should be tested and the breakpoint of ciprofloxacin established by MENSURA applied, instead of that of the NCCLS for these strains.