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Patients with chronic obstructive pulmonary disease (COPD) exhibit frequent acute exacerbations (AE). The objectives of this study were first to evaluate the prevalence of pathogens associated to these episodes by combining conventional bacteriology and multiplex viral and bacterial PCR assays in sputum specimens, and second to determine whether C-reactive protein (CRP) value and clinical outcome could be influenced by the type of microbial agent(s) recovered from these samples. A cohort of 84 Tunisian patients hospitalized at the emergency room for AECOPD was investigated prospectively for the semi-quantitative detection of bacteria by conventional culture (the threshold of positivity was of 10

Volume 9, issue 6, e02112-18, 2018, https://doi.org/10.1128/mBio.02112-18. In Data Set S1, the Raw Data Accession and Experiment Accession numbers of 516 samples were interchanged. The numbers have been corrected, and the file has been replaced online. In the main text, the isolates were referred to using the descriptor “sample” from Data Set S1, not any of the corrected accession numbers. Therefore, this change does not affect the main text of the article in any way. Nonetheless, we apologize to the readers for any inconvenience caused by this error.

Background Typhoid fever, caused by Salmonella Typhi, follows a fecal-oral transmission route and is a major global public health concern, especially in developing countries like Bangladesh. Increasing emergence of antimicrobial resistance (AMR) is a serious issue; the list of treatments for typhoid fever is ever-decreasing. In addition to IncHI1-type plasmids, Salmonella genomic island (SGI) 11 has been reported to carry AMR genes. Although reports suggest a recent reduction in multidrug resistance (MDR) in the Indian subcontinent, the corresponding genomic changes in the background are unknown. Results Here, we assembled and annotated complete closed chromosomes and plasmids for 73 S. Typhi isolates using short-length Illumina reads. S. Typhi had an open pan-genome, and the core genome was smaller than previously reported. Considering AMR genes, we identified five variants of SGI11, including the previously reported reference sequence. Five plasmids were identified, including the new plasmids pK91 and pK43; pK43and pHCM2 were not related to AMR. The pHCM1, pPRJEB21992 and pK91 plasmids carried AMR genes and, along with the SGI11 variants, were responsible for resistance phenotypes. pK91 also contained qnr genes, conferred high ciprofloxacin resistance and was related to the H58-sublineage Bdq, which shows the same phenotype. The presence of plasmids (pHCM1 and pK91) and SGI11 were linked to two H58-lineages, Ia and Bd. Loss of plasmids and integration of resistance genes in genomic islands could contribute to the fitness advantage of lineage Ia isolates. Conclusions Such events may explain why lineage Ia is globally widespread, while the Bd lineage is locally restricted. Further studies are required to understand how these S. Typhi AMR elements spread and generate new variants. Preventive measures such as vaccination programs should also be considered in endemic countries; such initiatives could potentially reduce the spread of AMR. Electronic supplementary material The online version of this article (10.1186/s12864-019-5916-6) contains supplementary material, which is available to authorized users.

Typhoid fever, caused by Salmonella enterica serovar Typhi, is responsible for an estimated burden of approximately 17 million new episodes per year worldwide. Adequate and timely antimicrobial treatment invariably cures typhoid fever. The increasing antimicrobial resistance (AMR) of S. Typhi severely limits the treatment options. We studied whole-genome sequences (WGS) of 536 S. Typhi isolates collected in Bangladesh between 1999 and 2013 and compared those sequences with data from a recent outbreak in Pakistan and a laboratory surveillance in Nepal. The analysis suggests that multiple ancestral origins of resistance against ciprofloxacin and ceftriaxone are present in three countries. Such independent genetic events and subsequent dissemination could enhance the risk of a rapid global spread of these highly resistant clones. Given the current treatment challenges, vaccination seems to be the most appropriate short-term intervention to reduce the disease burden of typhoid fever at a time of increasing AMR., Typhoid fever, caused by Salmonella enterica serovar Typhi, is a global public health concern due to increasing antimicrobial resistance (AMR). Characterization of S. Typhi genomes for AMR and the evolution of different lineages, especially in countries where typhoid fever is endemic such as Bangladesh, will help public health professionals to better design and implement appropriate preventive measures. We studied whole-genome sequences (WGS) of 536 S. Typhi isolates collected in Bangladesh during 1999 to 2013 and compared those sequences with data from a recent outbreak in Pakistan reported previously by E. J. Klemm, S. Shakoor, A. J. Page, F. N. Qamar, et al. (mBio 9:e00105-18, 2018, https://doi.org/10.1128/mBio.00105-18), and a laboratory surveillance in Nepal reported previously by C. D. Britto, Z. A. Dyson, S. Duchene, M. J. Carter, et al. [PLoS Negl. Trop. Dis. 12(4):e0006408, 2018, https://doi.org/10.1371/journal.pntd.0006408]. WGS had high sensitivity and specificity for prediction of ampicillin, chloramphenicol, co-trimoxazole, and ceftriaxone AMR phenotypes but needs further improvement for prediction of ciprofloxacin resistance. We detected a new local lineage of genotype 4.3.1 (named lineage Bd) which recently diverged into a sublineage (named Bdq) containing qnr genes associated with high-level ciprofloxacin resistance. We found a ceftriaxone-resistant isolate with the blaCTX-M-15 gene and a genotype distinct from the genotypes of extensively drug-resistant (XDR) isolates from Pakistan. This result suggests a different source and geographical origin of AMR. Genotype 4.3.1 was dominant in all three countries but formed country-specific clusters in the maximum likelihood phylogenetic tree. Thus, multiple independent genetic events leading to ciprofloxacin and ceftriaxone resistance took place in these neighboring regions of Pakistan, Nepal, and Bangladesh. These independent mutational events may enhance the risk of global spread of these highly resistant clones. A short-term global intervention plan is urgently needed.

International audience; An unexplained increase in the incidence of parapneumonic empyema (PPE) in pneumonia cases has been reported in recent years. The present study investigated the genetic and biological specifications of new isolates of torque teno mini virus (TTMV) detected in pleural effusion samples from children hospitalised for severe pneumonia with PPE. A pathogen discovery protocol was applied in undiagnosed pleural effusion samples and led to the identification of three new isolates of TTMV (TTMV-LY). Isolated TTMV-LY genomes were transfected into A549 and human embryonic kidney 293T cells and viral replication was assessed by quantitative real-time PCR and full-length genome amplification. A549 cells were further infected with released TTMV-LY virions and the induced-innate immune response was measured by multiplex immunoassays. Genetic analyses of the three TTMV-LY genomes revealed a classic genomic organisation but a weak identity (

Bronchiolitis is a major cause of morbidity and mortality in early childhood worldwide. The presence of more than one pathogen may influence the natural history of acute bronchiolitis in infants.To investigate the relevance of dual viral infection in infants with severe bronchiolitis hospitalized in a short-term unit compared with those in a pediatric intensive care unit (PICU).One hundred eighty infants1 year old hospitalized with bronchiolitis in a short-term unit (n = 92) or admitted to the PICU (n = 88) during 2 consecutive winter seasons 2003/2004 and 2004/2005 were evaluated. Molecular biology and standard methods were used to diagnose human respiratory viruses in nasal/throat swabs and nasal aspirates. Clinical data related to host factors and viral prevalence were compared among infants requiring or not PICU support.A viral agent was identified in 96.1% of infants with bronchiolitis. Respiratory syncytial virus (70.6% and 73.6%, respectively in the short-term unit and PICU) and rhinovirus (18.5% and 25.3%, respectively in the short-term unit and PICU) were the main detected respiratory viruses in infants hospitalized in both units. No significant difference in viral prevalence was observed between the populations studied. From multivariate analysis, infants with coinfections were 2.7 times (95% CI: 1.2-6.2) more at risk for PICU admission than those with a single infection. Respiratory syncytial virus and rhinovirus were the viruses most frequently identified in mixed infections in infants hospitalized with bronchiolitis.Dual viral infection is a relevant risk factor for the admission of infants with severe bronchiolitis to the PICU.

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.

Background It is estimated that Lao People’s Democratic Republic (Lao PDR) ranks fifth among the seven countries most affected by TB in the WHO Western Pacific Region. However, because of late implementation of mycobacterial culture, no study on resistance to anti-TB drugs had been performed yet. The objective of this study was to document drug resistance rate among patients hospitalized for pulmonary TB in threeprovinces of Lao PDR. Methods A cross-sectional study was conducted in three sites, one central and two regional hospitals, from April to November 2010. For each TB suspected patient sputum smear microscopy and culture on Lowenstein-Jensen media were performed. GenoType® MTBDRplus assay was used to test the susceptibility to isoniazid (INH) and rifampicin (RMP), GenoType® MTBDRsl for second-line drugs and GenoType® Mycobacterium CMAS for non-tuberculous mycobacteria (NTM). Results Out of 104 positive culture on Lowenstein-Jensen, 87 (83.6%) were M. tuberculosis and 17 (16.4%) were NTM. Of 73 new TB cases, 5 isolates (6.8%) were resistant to INH. Of 14 previously treated cases, 2 isolates (14.3%) were resistant to INH and one isolate was XDR. Conclusion Despite an overall rate of resistance still moderate, the frequency of mutations conferring INH monoresistance and identification of the first strain of XDR require strengthening surveillance of drug resistant tuberculosis in Lao PDR.

In order to establish the taxonomic value of 16S rRNA and 23S rRNA for distinguishing Listeria species, the complete 23S rRNA sequences for all Listeria species were determined by using the type strains. We designed and experimentally validated a universal 23S rRNA sequencing method, which included PCR amplification of the rDNA gene and direct cycle sequencing of the amplicon with eubacterial primers. The results of our sequence comparison indicated that the genus Listeria can be divided into two subgroups; one subgroup is composed of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri, whereas the other subgroup includes Listeria grayi subsp. grayi and Listeria grayi subsp. murrayi. A phylogenetic analysis revealed that these species diverged recently. These results are consistent with 16S rRNA sequence analysis data. For application purposes, one 16S rRNA region that can be sued to distinguish each Listeria species except L. Monocytogenes and L. innocua has been described. In this study we found four 23S rRNA signature regions which, when used in combination, can be used to distinguish the species.

The epidemiology of integron-mediated antibiotic-resistant genes in clinical enterobacteria from a single location was investigated. Forty-nine isolates (kindly provided by Dr. D. Sirot, Clermont-Ferrand, France) were selected for transferable resistance to aminoglycosides or to other antibiotics. Total DNA prepared from these strains was screened for the presence of conserved segments of integrons by PCR. The nature and frequency of inserted resistance gene cassettes were determined by direct nucleotide sequencing and were related to the resistances expressed by the strain. Integron hot-spots were present in 59% of the strains from 6 species, in either one or two copies. For amplicons sequenced, one or two antibiotic-resistant genes were found in various combinations, and were always expressed at the phenotypic level. They included the aminoglycoside resistance genes ant(3")-Ia and aac(6')-Ib (75%), as well as dhfr-I,-VII (21.4%) and blaOXA-1 (3.6%). Almost half of the transferable resistance to aminoglycosides (53%) was mediated by integron hot-spots in strains characterized at the nucleotide level. The proportion rose to 100% for the AAC(6')-I resistance profile. This study emphasizes the important contribution of integrons to aminoglycoside resistance within enterobacteria from a clinical setting.