Yersinia pestis: the Natural History of Plague.

The Gram-negative bacterium Yersinia pestis is responsible for deadly plague, a zoonotic disease established in stable foci in the Americas, Africa, and Eurasia. Its persistence in the environment relies on the subtle balance between Y. pestis-contaminated soils, burrowing and nonburrowing mammals exhibiting variable degrees of plague susceptibility, and their associated fleas. Transmission from one host to another relies mainly on infected flea bites, inducing typical painful, enlarged lymph nodes referred to as buboes, followed by septicemic dissemination of the pathogen. In contrast, droplet inhalation after close contact with infected mammals induces primary pneumonic plague. Finally, the rarely reported consumption of contaminated raw meat causes pharyngeal and gastrointestinal plague. Point-of-care diagnosis, early antibiotic treatment, and confinement measures contribute to outbreak control despite residual mortality. Mandatory primary prevention relies on the active surveillance of established plague foci and ectoparasite control. Plague is acknowledged to have infected human populations for at least 5,000 years in Eurasia. Y. pestis genomes recovered from affected archaeological sites have suggested clonal evolution from a common ancestor shared with the closely related enteric pathogen Yersinia pseudotuberculosis and have indicated that ymt gene acquisition during the Bronze Age conferred Y. pestis with ectoparasite transmissibility while maintaining its enteric transmissibility. Three historic pandemics, starting in 541 AD and continuing until today, have been described. At present, the third pandemic has become largely quiescent, with hundreds of human cases being reported mainly in a few impoverished African countries, where zoonotic plague is mostly transmitted to people by rodent-associated flea bites.

Preventing contamination of PCR-based multiplex assays including the use of a dedicated biosafety cabinet.

We retrospectively investigated cases of false-positive diagnoses using the BIOFIRE® FilmArray® meningitis/encephalitis (ME) panel to measure the impact of using a dedicated biosafety cabinet combined with preventive measures to reduce the prevalence of false-positive diagnoses due to pre-analytical in-laboratory contamination. False-positive results were identified by reviewing clinical data, biological parameters and cytology results of cerebrospinal fluid (CSF) samples showing discrepant results between the FilmArray ME panel and routine PCR assays. A total of 327 CSF were analysed over 16 weeks in point-of-care (POC) A and B, over two 8-week periods, periods 1 and 2. The analysis yielded 30 (9·17%) detection of at least one pathogen including 21/30 (70%) viruses and 9/30 (30%) bacteria. During period 1, POC-A and POC-B manipulated CSF under a non-dedicated hood featuring laminar flow, whereas during period 2, CSFs were manipulated under a dedicated biosafety cabinet without any airflow in POC-A. During period 1, false positives were detected in 3/114 CSF (2·63%) in POC-A and 1/36 (2·77%) in POC-B (P = 0·97); during period 2, false positives were detected in 0/139 CSF (0%) in POC-A and 1/38 (2·63%) in POC-B (P = 0·23). All false positives were bacterial. The use of a dedicated cabinet without ventilation along with preventive measures during period 2 in POC-A significantly reduced the number of false-positive results (P = 0·05). Preventive measures described in this study can mitigate false positives when using PCR-based multiplex assays such as the BIOFIRE FilmArray ME Panel for the diagnosis of meningitis and other infectious diseases.

Detection of methanogens in peri-appendicular abscesses: Report of four cases.

The aetiology of appendicular abscess is predominantly microbial with aerobic and anaerobic bacteria from gut flora. In this study, by using specific laboratory tools, we co-detected Methanobrevibacter oralis and Methanobrevibacter smithii among a mixture of enterobacteria including Escherichia coli, Enterococcus faecium and Enterococcus avium in four unrelated cases of postoperative appendiceal abscesses. These unprecedented observations raise a question on the role of methanogens in peri-appendicular abscesses, supporting antibiotics as an alternative therapeutic option for appendicitis, including antibiotics active against methanogens such as metronidazole or fusidic acid.

The diversity of mycolactone-producing mycobacteria.

Mycolactone-producing mycobacteria (MPM) form an intriguing group of environmental opportunistic pathogens of mammals and human patients in whom they cause cutaneous and subcutaneous ulcers known as "Buruli ulcer" when they occur in humans. We reviewed whole genome sequence data and ecological and phenotypic characteristics from 44 MPMs and closely related Mycobacterium marinum. This analysis indicated that all the 24 M. marinum isolates were delineated into seven taxa and our comprehensive, polyphasic taxonomic approach led to the proposal of delineating M. marinum genomospecies, 01-07. Likewise, 20 MPMs isolates were delineated into seven additional M. ulcerans genomospecies, 01-07. A taxonomic card explaining the ecology, hosts of isolation and the plasmid harboured is provided for each taxon.

Digestive tract methanodrome: Physiological roles of human microbiota-associated methanogens.

Methanogens are the archaea most commonly found in humans, in particular in the digestive tract and are an integral part of the digestive microbiota. They are present in humans from the earliest moments of life and represent the only known source of methane production to date. They are notably detected in humans by microscopy, fluorescent in situ hybridization, molecular biology including PCR-sequencing, metagenomics, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and culture. Methanogens present in the human digestive tract play major roles, in particular the use of hydrogen from the fermentation products of bacteria, thus promoting digestion. They are also involved in the transformation of heavy metals and in the use of trimethylamine produced by intestinal bacteria, thus preventing major health problems, in particular cardiovascular diseases. Several pieces of evidence suggest their close physical contacts with bacteria support symbiotic metabolism. Their imbalance during dysbiosis is associated with many pathologies in humans, particularly digestive tract diseases such as Crohn's disease, ulcerative colitis, diverticulosis, inflammatory bowel disease, irritable bowel syndrome, colonic polyposis, and colorectal cancer. There is a huge deficit of knowledge and partially contradictory information concerning human methanogens, so much remains to be done to fully understand their physiological role in humans. It is necessary to develop new methods for the identification and culture of methanogens from clinical samples. This will permit to isolate new methanogens species as well as their phenotypic characterization, to explore their genome by sequencing and to study the population dynamics of methanogens by specifying in particular their exact role within the complex flora associated with the mucous microbiota of human.

Methanogens as emerging pathogens in anaerobic abscesses.

Methanogens are strictly anaerobic archaea metabolising by-products of bacterial fermentation into methane by using three known metabolic pathways, i.e. the reduction of carbon dioxide, the fermentation of acetate or the dismutation of methanol or methylamines. Methanogens described in human microbiota include only Euryarchaeota, i.e. Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter arbophilus, Methanobrevibacter massiliensis, Methanomassiliicoccus luminyensis, Methanosphaera stadtmanae and Ca. Methanomethylophilus alvus and Ca. Methanomassiliicoccus intestinalis. Methanogens are emerging pathogens associated with brain and muscular abscesses. They have been implicated in dysbiosis of the oral microbiota, periodontitis and peri-implantitis. They have also been associated with dysbiosis of the digestive tract microbiota linked to metabolic disorders (anorexia, malnutrition and obesity) and with lesions of the digestive tract (colon cancer). Their detection in anaerobic pus specimens and oral and digestive tract specimens relies on microscopic examination by fluorescence in situ hybridisation, specific DNA extraction followed by polymerase chain reaction (PCR)-based amplification of the 16S rRNA and mcrA gene fragments and isolation and culture in the supporting presence of hydrogen-producing bacteria. Diagnostic identification can be performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and can be further completed by genotyping through multi-spacer sequencing and, ultimately, whole genome sequencing (WGS). Ornidazole derivatives, fusidic acid and rifampicin are the compounds to be included in in vitro susceptibility testing to complete the clinical workflow. Clinical microbiology laboratories should work toward developing cheap and easy protocols for the routine detection and identification of methanogens in selected specimens in order to refine the diagnosis of infections, as well as to expand the knowledge about this group of intriguing microorganisms.

Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach.

Methanobrevibacter smithii is the main human methanogen almost always found in the digestive tract of adults. Yet, the age at which M. smithii establishes itself as part of the developing intestinal microflora remains unknown. In order to gain insight into this, we developed a polyphasic approach, including microscopic observation by fluorescence in situ hybridisation, polymerase chain reaction (PCR) sequencing detection, identification and culture, to isolate and genotype M. smithii in one-day-old newborns' gastric juice specimens. In the presence of negative controls, 50/50 (100%) prospectively analysed newborn gastric juice specimens were PCR-positive for methanogens, all identified as M. smithii by sequencing. We succeeded in cultivating M. smithii in 35/50 (70%) newborn gastric juice specimens, while 15/50 specimens remained sterile. Further, M. smithii was observed by direct microscopic investigation using fluorescence in situ hybridisation. Multispacer sequence typing found one of seven different genotypes per specimen, these genotypes having all been previously described in adult human stools. Methanobrevibacter smithii is an early inhabitant of the human stomach, colonising the gastric mucosa just after birth, and the mother's gut microbiota is a probable source of colonisation.

Direct matrix-assisted laser desorption ionisation time-of-flight mass spectrometry identification of mycobacteria from colonies.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) identification of mycobacteria requires a standard acetonitrile/formic acid pre-MALDI-TOF-MS. We prospectively compared this standard protocol with direct deposit with matrix for the identification of mycobacteria cultured on solid media. We first verified that Mycobacterium tuberculosis was killed after it was mixed with matrix. Then, 111 Mycobacterium isolates previously identified by partial rpoB gene sequencing were tested in parallel by the two protocols. An identification score >1.7 was obtained in 86/111 (77.5 %) isolates after protein extraction versus 97/111 (87.4 %) isolates after direct deposit (p = 0.039, Chi-squared test). In a third step, we determined that direct deposit achieved identification for as few as 2.104 M. tuberculosis organisms. In a fourth step, we evaluated direct deposit of one colony for 116 solid medium-cultured clinical isolates finally identified as representative of 12 species (63.8 % M. tuberculosis). For 114/116 (98.3 %) isolates with an identification score >1.2, the MALDI-TOF-MS identification was in complete agreement with the reference rpoB gene sequencing identification. One isolate with a MALDI-TOF-MS identification score of 1.22 for M. fortuitum was identified as M. avium by partial rpoB gene sequencing. One other isolate with a MALDI-TOF-MS identification score of 1.22 for M. tuberculosis was identified as M. tuberculosis by genotyping. All the original MALDI-TOF-MS spectra reported here have been deposited in a public database. Direct deposit of one colony on a MALDI-TOF-MS plate allows for an accurate identification of mycobacteria for an identification score >1.3.

Aerobic culture of methanogenic archaea without an external source of hydrogen.

Culturing methanogenic archaea is fastidious, expensive, and requires an external source of hydrogen and carbon dioxide. Until now, these microorganisms have only been cultivated under strictly anaerobic conditions. We previously developed a single versatile culture medium containing sugars and anti-oxydants for cultivating all human known methanogens. Performing aerobic cultures in the presence of Bacteroides thetaiotaomicron, which produces hydrogen, allows for cultivation of Methanobrevibacter smithii which itself produces methane. To obtain colonies, we cultivated M. smithii in an agar plate in the upper part of a double chamber flask with a liquid culture of B. thetaiotaomicron in the lower compartment. We subsequently cultured four other methanogenic species for the first time and successfully isolated 13 strains of M. smithii and nine strains of Methanobrevibacter oralis from 100 stools and 45 oral samples. This procedure allows aerobic isolation and antibiotic susceptibility testing. This changes the ability to routinely study methanogens, which have been neglected in clinical microbiology laboratories and may be useful for biogas production.

A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens.

The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causative Mycobacterium tuberculosis bacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21 M. tuberculosis isolates at 10(5), 10(3), and 10 CFU yielded colonies in 5.7 ± 1.5 days, 7.6 ± 0.8 days, and 10.8 ± 1.7 days versus 1.5 ± 0.4 days, 3.5 ± 0.6 days, and 4.9 ± 1 days in MOD9 (P < 0.05, Student's t test). Further, the time to detectable growth of M. tuberculosis was measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P = 0.11, Fisher's exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P = 0.5195, Fisher's exact test). All together, eight M. tuberculosis isolates were cultured solely from chlorhexidine-MOD9, and two M. tuberculosis isolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ± 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ± 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P < 0.05, Student's t test). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery of M. tuberculosis.

Genetic variants of dental plaque Methanobrevibacter oralis.

Methanobrevibacter oralis is the major methanogenic archaea found in the oral cavity. It has been implicated in periodontitis, including the severe form. It is unknown whether certain M. oralis genetic variants are associated with severe periodontitis. Here, we developed multispacer sequence typing (MST) as a sequencing-based genotyping method for the assessment of M. oralis. The sequencing of four intergenic spacers from a collection of 17 dental plaque M. oralis isolates obtained from seven individuals revealed 482 genetic polymorphisms, including 401 single nucleotide polymorphisms (83.2 %), 55 deletions (11.4 %) and 26 insertions (5.4 %). Concatenation of the four spacers yielded nine genotypes, which were clustered into six groups with an index of discrimination of 0.919. One periodontitis patient may have harboured up to three genetic variants of M. oralis, revealing the previously unknown diversity of this archaea. MST will allow for the study of the dynamics of M. oralis populations, including inter-individual transmission and any correlations with the severity of periodontitis.

Complementarity between targeted real-time specific PCR and conventional broad-range 16S rDNA PCR in the syndrome-driven diagnosis of infectious diseases.

Molecular tools have shown an added value in the diagnosis of infectious diseases, in particular for those caused by fastidious intracellular microorganisms, or in patients receiving antibiotics before sampling. If 16S rDNA amplification had been gradually implemented in microbiology laboratories, specific real-time polymerase chain reaction (PCR) would have permitted an increase in the sensitivity of molecular methods and a reduction of contamination. Herein, we report our experience in the diagnosis of infectious diseases over two years, during which 32,948 clinical samples from 18,056 patients were received from France and abroad. Among these samples, 81,476 PCRs were performed, of which 1,192 were positive. Molecular techniques detected intracellular microorganisms in 31.3 % of respiratory samples, 27.8 % of endocarditis samples and 51.9 % of adenitis samples. Excluding intracellular bacteria, 25 % of the positive samples in this series were sterile in culture. Conventional broad-range PCR permitted the identification of fastidious and anaerobic microorganisms, but specific real-time PCR showed a significant superiority in the diagnosis of osteoarticular infections, in particular for those caused by Kingella kingae and Staphylococcus aureus, and for endocarditis diagnosis, specifically when Streptococcus gallolyticus and Staphylococcus aureus were involved. The sensitivity of conventional broad-range PCR was 62.9 % concerning overall diagnoses for which both techniques had been performed. These findings should lead microbiologists to focus on targeted specific real-time PCR regarding the clinical syndrome. Finally, syndrome-driven diagnosis, which consists of testing a panel of microorganisms commonly involved for each syndrome, permitted the establishment of 31 incidental diagnoses.

Colonization of Hospital Water Networks by Gemmata massiliana, a New Planctomycetes Bacterium.

Planctomycetes have been isolated from various hydric environments. These fastidious bacteria are overlooked by routine 16S rRNA gene-based PCR detection in hospital laboratories, and their presence has not been documented in the health-care environment. Using a specific culture protocol, we recently isolated a new, non-filterable Planctomycetes species, Gemmata massiliana, from one hospital water network. The goal of the study was to monitor the presence of G. massiliana in two hospital water networks. We developed a G. massiliana-specific real-time PCR system and monitored the presence of the Planctomycetes for 12 months in two hospital water networks, in filtered water collected at the intensive care unit and in non-filtered water collected from dental chairs, tanks, and usage points. Four of 180 (2.2%) filtered water samples tested positive versus 23 of 204 (11.3%) non-filtered points (p < 0.05), including 18 of 128 (14.1%) dental chairs, 3 of 51 (5.9%) usage points, and two of 25 (8%) tank specimens. There was no significant difference in the prevalence of G. massiliana between the two hospitals (p > 0.05). However, this organism was detected significantly more frequently during April and September than the 10 other months. Because G. massiliana is deeply entrenched in the hospitalized patient's environment, evaluating this organism as a new opportunistic, health-care-associated pathogen is warranted.

Planctomycetes DNA in febrile aplastic patients with leukemia, rash, diarrhea, and micronodular pneumonia.

We found Planctomycetes DNA in 2 out of 100 blood samples from patients suffering from leukemia with neutropenia induced by chemotherapy, as well as fever, rash, pneumonia, and diarrhea. Antibiotic-resisting Planctomycetes may be pathogenic in these patients.

Disseminated Mycobacterium lentiflavum responsible for hemophagocytic lymphohistocytosis in a man with a history of heart transplantation.

Mycobacterium lentiflavum is a nontuberculous, slowly growing mycobacterium usually recognized as a contaminant. Here, we report a case of disseminated M. lentiflavum infection responsible for hemophagocytic lymphohistocytosis in a heart-transplanted man.

Looking in amoebae as a source of mycobacteria.

Mycobacteria exhibit various relationships with amoebae, ranging from the killing of one partner by the other one, to amoebae hosting mycobacteria in trophozoites and cysts. This observation indicates that poorly described biological factors affect the relationships, including mycobacterial cell-wall glycolipids and the size of the mycobacteria. Experimental observations indicate that a majority of environmental, opportunistic mycobacteria but also obligate pathogens including Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium ulcerans are inter-amoebal organisms. Amoebae may give opportunities for genetic exchanges between mycobacteria, sympatric intra-amoebal organisms and the amoebae themselves. Amoebae clearly protect opportunistic mycobacterial pathogens during their environmental life but their role for obligate mycobacterial infection remains to be established. Accordingly, water was the source for emerging, community-acquired and health care-associated infection with amoeba-resisting mycobacteria of the Mycobacterium avium, Mycobacterium abscessus and Mycobacterium fortuitum groups, among others. Amoebae are organisms where mycobacteria can be found and, accordingly, amoeba co-culture can be used for the isolation of mycobacteria from environmental and clinical specimens. Looking in amoebae may help recovering new species of mycobacteria.

Paleoserological detection of Coronavirus antigens in dental calculus of human remains dating from the beginning of the 19th century, French Ardennes.

OBJECTIVE: Vanishing viral RNA restricts our ability to detect ancient pathogens, so, we used paleo serological approaches to trace the dynamics of the Coronavirus in ancient populations. MATERIALS AND METHODS: We investigated 10 ancient dental calculus samples collected from a cemetery dated to the beginning of the 19th century and excavated in Charleville-Mézières. After paleoserum samples were extracted from dental calculus, paleoserology using mini-line-blot incorporating one alpha-Coronavirus (Coronavirus 229 E) and two beta-Coronavirus (Coronavirus OC 43, SARS-CoV-2) antigens and controls was completed by an automated Western blotting assay. RESULTS: Once appropriate controls had validated the data, mini-line-blot detected antibodies against the two beta-Coronavirus antigens in individuals US1300 and US1339, automated Western blotting confirming one beta-Coronavirus antigen for individual US1300 and an additional individual US1326. DISCUSSION: Combing mini-line blot and automated Western blot assays made it possible to detect immunoreactive immunoglobulin tracing circulation of Coronavirus in France at the very beginning of the 19th century.

Gastric aspiration is not necessary for the diagnosis of pulmonary tuberculosis.

Despite recommendations, gastric aspirate collected by invasive nasogastric aspiration is still routinely used for the direct detection of Mycobacterium tuberculosis in our institution. Reviewing 82 patients with culture-proven respiratory tuberculosis over a 28-month period, we observed no patient diagnosed solely by gastric aspirate analysis. Moreover, the diagnosis yield of gastric aspirate (60 %) did not significantly differ from that of stool specimen (64 %). These data confirm that gastric aspirate is no longer useful for the diagnosis of respiratory tuberculosis contrary to stool specimen.