Jean Marc Rolain

Ongoing Revolution in Bacteriology: Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

BACKGROUNDnMatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic.nnnMETHODSnWe prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured.nnnRESULTSnOf 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification.nnnCONCLUSIONSnMALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising > or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.

Rickettsial infections and fever, Vientiane, Laos.

Rickettsia spp. are an underrecognized cause of undifferentiated febrile illness.

Culture of previously uncultured members of the human gut microbiota by culturomics

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism1. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms2. We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization–time of flight and 16S rRNA for identification2. Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies3–5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.

Rickettsia slovaca and R. raoultii in tick-borne Rickettsioses.

Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), is defined as the association of a tick bite, an inoculation eschar on the scalp, and cervical adenopathies. We identified the etiologic agent for 65% of 86 patients with TIBOLA/DEBONEL as either Rickettsia slovaca (49/86, 57%) or R. raoultii (7/86, 8%).

New Delhi metallo-beta-lactamase (NDM-1): towards a new pandemia?

During the past decade the increase of antibiotic resistance in Enterobacteriaceae has become a major concern worldwide. Although beta-lactams have been widely used as the mainstay of treatment for severe infections due to these bacteria, with carbapenems often representing last-resource drugs, carbapenem resistance due to acquired carbapenemases has emerged and spread worldwide since the early 2000s, being even more worrisome for public health because these bacteria are a common source of hospital-acquired infections. Carbapenemases have been now studied in depth, and widely differ from one another, including enzymes from class B (metallo-beta-lactamases, MBLs), class A and class D (serine carbapenemases) [1]. The most prevalent carbapenemase so far in Enterobacteriaceae is the KPC-type class-A carbapenemase, which has been found in Klebsiella pneumonia, especially in the United States, Asia, the United Kingdom, Israel and southern Europe [2]. Interestingly, acquired carbapenemases have been mainly restricted to geographical areas and to specific bacterial species, and outbreaks as well as spread in other countries have been often associated with imported cases from countries where the bacteria are endemic. Population mobility is known to be a main factor in globalization and spreading of antimicrobial drug-resistant organisms [3]. For example, the emergence of KPC-producing Enterobacteriaceae in the United States in 2001 [2] could be later associated with the emergence of travel-related outbreaks in other countries [1,4]. The New Delhi metallo-beta-lactamase (NDM-1) is a novel type of MBL named after the city of origin, which has been recently criticized, following a common practice with transferable MBLs since VIM-1 was named after Verona, Italy [5]. NDM-1 was first reported in 2009 in a Swedish patient of Indian origin, who travelled to New Delhi and acquired a urinary tract infection due to a carbapenem-resistant K. pneumoniae strain resistant to all antibiotics tested except colistin [6]. Faecal samples collected from this patient during his stay at the nursing home yielded an NDM-1 positive E. coli as well [6]. The NDM-1 encoding gene is located on different large plasmids (a 180-kb plasmid for K. pneumoniae and a 140-kb plasmid for E. coli) that are easily transferable to susceptible E. coli J53 at a high frequency [6]. These plasmids also harbour genes conferring resistance to almost all antibiotics, thus making their rapid dissemination in clinically relevant bacteria a serious threat for therapy. Following this first case, sporadic cases of infection due to NDM-1 positive bacteria have been detected, including an E. coli from blood cultures of a patient of Indian origin in the United Kingdom [7], three cases of Enterobacteriaceae isolates (one E. coli, one K. pneumoniae and one E. cloacae) in the United States from patients who received care in India [8], and three cases of Acinetobacter baumannii from New Delhi [9]. In the August issue of the journal The Lancet: Infectious Diseases, a multinational team reported the emergence and spread of 180 cases of patients infected by bacteria carrying the NDM-1, including 37 cases in the United Kingdom and 143 cases in various sites in Pakistan and India, thus suggesting a widespread dissemination [10]. Among these bacteria many different Enterobacteriaceae species were identified, including K. pneumonia, E. coli, E. cloacae, Proteus spp., Citrobacter freundii, K. oxytoca, M. morganii and Providencia spp. Most isolates remained susceptible to colistin and tigecycline, except those Enterobacteriaceae endowed with a natural resistance to these compounds such as M. morganii, Proteus spp. and Providencia spp. Most plasmids detected in these bacteria were easily transferable and capable of wide rearrangement, suggesting a widespread transmission and plasticity among bacterial populations. Interestingly, among the 25 patients detected in the UK, 17 patients had travelled to India or Pakistan within 1 year and 14 had been hospitalized in these countries showing a worldwide dissemination of a new ‘superbug’ from a local source in Asia [10]. Indeed, since August 2010, spreading and dissemination has occurred, with several cases being reported by national and international media from other countries in all continents, including the United States and Canada, Europe (Sweden, the United Kingdom, Austria, Belgium, France, Netherlands and Germany), Japan, Africa,

Ectoparasitism and vector-borne diseases in 930 homeless people from Marseilles

Abstract: Homeless people are particularly exposed to ectoparasites, but their exposure to arthropod-borne diseases has not been evaluated systematically. A medical team of 27 persons (7 nurses, 6 infectious disease residents or fellows, 2 dermatologists, and 12 infectious disease specialists) visited the 2 shelters in Marseilles, France, for 4 consecutive years. Homeless volunteers were interviewed, examined, and received care; and blood was sampled for cell counts and detection of bacteremia, antibodies to louse-borne (Rickettsia prowazekii, Bartonella quintana, and Borrelia recurrentis), flea-borne (R. typhi, R. felis), mite-borne (R. akari), and tick-borne (R. conorii) bacterial agents. We selected sex- and age-adjusted controls among healthy blood donors. Over 4 years, 930 homeless people were enrolled. Lice were found in 22% and were associated with hypereosinophilia (odds ratio, 5.7; 95% confidence intervals, 1.46-22.15). Twenty-seven patients (3%) with scabies were treated with ivermectin. Bartonella quintana was isolated from blood culture in 50 patients (5.3%), 36 of whom were treated effectively. The number of bacteremic patient increased from 3.4% to 8.4% (p = 0.02) over the 4 years of the study. We detected a higher seroprevalence to Borrelia recurrentis, R. conorii, and R. prowazekii antibodies in the homeless. Our study shows a high prevalence of louse-borne infections in the homeless and a high degree of exposure to tick-borne diseases and scabies. Despite effective treatment for Bartonella quintana bacteremia and the efforts made to delouse this population, Bartonella quintana remains endemic, and we found hallmarks of epidemic typhus and relapsing fever. The uncontrolled louse infestation of this population should alert the community to the possibility of severe re-emerging louse-borne infections.

Tropheryma whipplei in Children with Gastroenteritis

This bacterium may be an etiologic agent of gastroenteritis.

Genotyping reveals a wide heterogeneity of Tropheryma whipplei.

Tropheryma whipplei, the causative agent of Whipples disease, is associated with various clinical manifestations as well as an asymptomatic carrier status, and it exhibits genetic heterogeneity. However, relationships that may exist between environmental and clinical strains are unknown. Herein, we developed an efficient genotyping system based on four highly variable genomic sequences (HVGSs) selected on the basis of genome comparison. We analysed 39 samples from 39 patients with Whipples disease and 10 samples from 10 asymptomatic carriers. Twenty-six classic gastrointestinal Whipples disease associated with additional manifestations, six relapses of classic Whipples disease (three gastrointestinal and three neurological relapses), and seven isolated infections due to T. whipplei without digestive involvement (five endocarditis, one spondylodiscitis and one neurological infection) were included in the study. We identified 24 HVGS genotypes among 39 T. whipplei DNA samples from the patients and 10 T. whipplei DNA samples from the asymptomatic carriers. No significant correlation between HVGS genotypes and clinical manifestations of Whipples disease, or asymptomatic carriers, was found for the 49 samples tested. Our observations revealed a high genetic diversity of T. whipplei strains that is apparently independent of geographical distribution and unrelated to bacterial pathogenicity. Genotyping in Whipples disease may, however, be useful in epidemiological studies.

Bacteriophages and diffusion of genes encoding antimicrobial resistance in cystic fibrosis sputum microbiota

OBJECTIVESnThe cystic fibrosis (CF) airway is now considered the site of a complex microbiota, where cross-talking between microbes and lateral gene transfer are believed to contribute to the adaptation of bacteria to this specific environment and to the emergence of multidrug-resistant bacteria. The objective of this study was to retrieve and analyse specific sequences associated with antimicrobial resistance from the CF viromes database.nnnMETHODSnSpecific sequences from CF metagenomic studies related to the antibiotic and toxic compound resistance dataset were retrieved from the MG-RAST web site, assembled and functionally annotated for identification of the genes. Phylogenetic trees were constructed using a minimum parsimony starting tree topology search strategy.nnnRESULTSnOverall, we found 1031 short sequences in the CF virome putatively encoding resistance to antimicrobials versus only 3 reads in the non-CF virome dataset (Pu200a=u200a0.001). Among them, we could confidently identify 66 efflux pump genes, 15 fluoroquinolone resistance genes and 9 β-lactamase genes. Evolutionary relatedness determined using phylogenetic information demonstrates the different origins of these genes among the CF microbiota. Interestingly, among annotated sequences within CF viromes, we also found matching 16S rDNA sequences from Escherichia, Cyanobacteria and Bacteroidetes.nnnCONCLUSIONSnOur results suggest that phages in the CF sputum microbiota represent a reservoir of mobilizable genes associated with antimicrobial resistance that may spread in this specific niche. This phenomenon could explain the fantastic adaptation of CF strains to their niche and may represent a new potential therapeutic target to prevent the emergence of multidrug-resistant bacteria, which are responsible for most of the deaths in CF.

New insights into the antibacterial mechanism of action of squalamine

OBJECTIVESnAntimicrobial resistance is an increasingly life-threatening problem that emphasizes the need to develop new antibacterial agents. The in vitro antibacterial activity of squalamine, a natural aminosterol, has been previously demonstrated against multidrug-resistant bacteria and moulds. Although the antibacterial activity of squalamine was found to correlate with that of other drugs, such as colistin, against Gram-negative bacteria, the former was active against Gram-positive bacteria, which are resistant to colistin. In this work, we provide new insights into squalamines antibacterial mechanism of action compared with other known antibiotics.nnnMETHODSnWe evaluated squalamines antibacterial mechanism of action using the broth microdilution method for MIC determination and time-kill assays, transmission electron microscopy for morphological change studies, bioluminescence for ATP release measurements and fluorescence methods for membrane depolarization assays.nnnRESULTSnConcerning Gram-negative bacteria, squalamine, similar to colistin, required interaction with the negatively charged phosphate groups in the bacterial outer membrane as the first step in a sequence of different events ultimately leading to the disruption of the membrane. Conversely, squalamine exhibited a depolarizing effect on Gram-positive bacteria, which resulted in rapid cell death.nnnCONCLUSIONSnThe new insights into the mechanism of action of squalamine highlight the importance of aminosterols in the design of a new class of antibacterial compounds that could be used as disinfectants and detergents.