Population dynamics of plague vector fleas in an endemic focus: implications for plague surveillance.

Plague is a zoonotic vector-borne disease caused by the bacterium Yersinia pestis. In Madagascar, it persists in identified foci, where it is a threat to public health generally from September to April. A more complete understanding of how the disease persists could guide control strategies. Fleas are the main vector for transmission between small mammal hosts and humans, and fleas likely play a role in the maintenance of plague. This study characterized the dynamics of flea populations in plague foci alongside the occurrence of human cases. From 2018 to 2020, small mammals were trapped at sites in the central Highlands of Madagascar. A total of 2,762 small mammals were captured and 5,295 fleas were collected. The analysis examines 2 plague vector species in Madagascar (Synopsyllus fonquerniei and Xenopsylla cheopis). Generalized linear models were used to relate flea abundance to abiotic factors, with adjustments for trap location and flea species. We observed significant effects of abiotic factors on the abundance, intensity, and infestation rate by the outdoor-associated flea species, S. fonquerniei, but weak seasonality for the indoor-associated flea species, X. cheopis. A difference in the timing of peak abundance was observed between the 2 flea species during and outside the plague season. While the present study did not identify a clear link between flea population dynamics and plague maintenance, as only one collected X. cheopis was infected, the results presented herein can be used by local health authorities to improve monitoring and control strategies of plague vector fleas in Madagascar.

New Bacteriophages with Podoviridal Morphotypes Active against Yersinia pestis: Characterization and Application Potential.

Phages of highly pathogenic bacteria represent an area of growing interest for bacterial detection and identification and subspecies typing, as well as for phage therapy and environmental decontamination. Eight new phages-YpEc56, YpEc56D, YpEc57, YpEe58, YpEc1, YpEc2, YpEc11, and YpYeO9-expressing lytic activity towards Yersinia pestis revealed a virion morphology consistent with the Podoviridae morphotype. These phages lyse all 68 strains from 2 different sets of Y. pestis isolates, thus limiting their potential application for subtyping of Y. pestis strains but making them rather promising in terms of infection control. Two phages-YpYeO9 and YpEc11-were selected for detailed studies based on their source of isolation and lytic cross activity towards other Enterobacteriaceae. The full genome sequencing demonstrated the virulent nature of new phages. Phage YpYeO9 was identified as a member of the Teseptimavirus genus and YpEc11 was identified as a member of the Helsettvirus genus, thereby representing new species. A bacterial challenge assay in liquid microcosm with a YpYeO9/YpEc11 phage mixture showed elimination of Y. pestis EV76 during 4 h at a P/B ratio of 1000:1. These results, in combination with high lysis stability results of phages in liquid culture, the low frequency of formation of phage resistant mutants, and their viability under different physical-chemical factors indicate their potential for their practical use as an antibacterial mean.

Rodent control to fight plague: field assessment of methods based on rat density reduction.

Rodents represent a serious threat to food security and public health. The extent to which rodent control can mitigate the risk from rodent-borne disease depends on both the effectiveness of control in reducing rodent abundance and the impact on disease epidemiology. Focusing on a plague-endemic region of Madagascar, this study compared the effectiveness of 3 methods: live-traps, snap-traps, and rodenticides. Control interventions were implemented inside houses between May and October 2019. Tracking tiles monitored rodent abundance. Rodent fleas, the vector involved in plague transmission, were collected. Rodent populations consisted of Rattus rattus and Mus musculus. In terms of trap success, we found that our live-trap regime was more effective than snap-traps. While all 3 control strategies appeared to reduce in-house rodent activity in the short term, we found no evidence of a longer-term effect, with in-house rodent abundance in treated sites comparable to non-treatment sites by the following month. Endemic flea, Synopsyllus fonquerniei, is a key plague vector usually found on rats living outdoors. Although we found no evidence that its abundance inside houses increased following control, this may have been due to a lack of power caused by significant variation in S. fonquerniei abundance. The presence of S. fonquerniei in houses was more likely when S. fonquerniei abundance on outdoor rats was higher, which in turn correlated with high rat abundance. Our results emphasize that control strategies need to consider this connectivity between in-house rat-flea populations and the outdoor populations, and any potential consequences for plague transmission.

Genomic and RT-qPCR analysis of trimethoprim-sulfamethoxazole and meropenem resistance in Burkholderia pseudomallei clinical isolates.

BACKGROUND: Melioidosis is an endemic disease in southeast Asia and northern Australia caused by the saprophytic bacteria Burkholderia pseudomallei, with a high mortality rate. The clinical presentation is multifaceted, with symptoms ranging from acute septicemia to multiple chronic abscesses. Here, we report a chronic case of melioidosis in a patient who lived in Malaysia in the 70s and was suspected of contracting tuberculosis. Approximately 40 years later, in 2014, he was diagnosed with pauci-symptomatic melioidosis during a routine examination. Four strains were isolated from a single sample. They showed divergent morphotypes and divergent antibiotic susceptibility, with some strains showing resistance to trimethoprim-sulfamethoxazole and fluoroquinolones. In 2016, clinical samples were still positive for B. pseudomallei, and only one type of strain, showing atypical resistance to meropenem, was isolated. PRINCIPAL FINDINGS: We performed whole genome sequencing and RT-qPCR analysis on the strains isolated during this study to gain further insights into their differences. We thus identified two types of resistance mechanisms in these clinical strains. The first one was an adaptive and transient mechanism that disappeared during the course of laboratory sub-cultures; the second was a mutation in the efflux pump regulator amrR, associated with the overexpression of the related transporter. CONCLUSION: The development of such mechanisms may have a clinical impact on antibiotic treatment. Indeed, their transient nature could lead to an undiagnosed resistance. Efflux overexpression due to mutation leads to an important multiple resistance, reducing the effectiveness of antibiotics during treatment.

Case Report of an Injectional Anthrax in France, 2012.

(1) Background: Bacillus anthracis is a spore-forming, Gram-positive bacterium causing anthrax, a zoonosis affecting mainly livestock. When occasionally infecting humans, B. anthracis provokes three different clinical forms: cutaneous, digestive and inhalational anthrax. More recently, an injectional anthrax form has been described in intravenous drug users. (2) Case presentation: We report here the clinical and microbiological features, as well as the strain phylogenetic analysis, of the only injectional anthrax case observed in France so far. A 27-year-old patient presented a massive dermohypodermatitis with an extensive edema of the right arm, and the development of drug-resistant shocks. After three weeks in an intensive care unit, the patient recovered, but the microbiological identification of B. anthracis was achieved after a long delay. (3) Conclusions: Anthrax diagnostic may be difficult clinically and microbiologically. The phylogenetic analysis of the Bacillus anthracis strain PF1 confirmed its relatedness to the injectional anthrax European outbreak group-II.

Three New Cases of Melioidosis, Guadeloupe, French West Indies.

Melioidosis has been detected in the Caribbean, and an increasing number of cases has been reported in the past few decades, but only 2 cases were reported in Guadeloupe during the past 20 years. We describe 3 more cases that occurred during 2016-2017 and examine arguments for increasing endemicity.

Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations.

Melioidosis in the Western Indian Ocean and the Importance of Improving Diagnosis, Surveillance, and Molecular Typing.

Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an infectious disease of humans or animals, and the specific environmental conditions that are present in western Indian Ocean islands are particularly suitable for the establishment/survival of B. pseudomallei. Indeed, an increasing number of new cases have been reported in this region (Madagascar, Mauritius, Réunion (France), and Seychelles, except Comoros and Mayotte (France)), and are described in this review. Our review clearly points out that further studies are needed in order to investigate the real incidence and burden of melioidosis in the western Indian Ocean and especially Madagascar, since it is likely to be higher than currently reported. Thus, research and surveillance priorities were recommended (i) to improve awareness of melioidosis in the population and among clinicians; (ii) to improve diagnostics, in order to provide rapid and effective treatment; (iii) to implement a surveillance and reporting system in the western Indian Ocean; and (iv) to investigate the presence of B. pseudomallei in environmental samples, since we have demonstrated its presence in soil samples originating from the yard of a Madagascan case.

Clinical isolates of Escherichia coli solely resistant to mecillinam: prevalence and epidemiology.

In routine susceptibility testing of Gram-negative bacteria, a particular resistance phenotype was observed: an Escherichia coli isolate from a urine sample exhibited resistance solely to mecillinam (MEC) but was fully susceptible to other ß-lactam antibiotics (MEC-R-BL-S). The objectives as this study were to determine the prevalence of this phenotype and to describe the phenotype, molecular epidemiology and genetic background. Between 1 January 2014 and 31 January 2016, MEC-R-BL-S E. coli isolates from urine were collected and genes previously reported as mostly involved in MEC resistance were analysed. The genetic relatedness among isolates was investigated by repetitive element sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). Ten MEC-R-BL-S isolates were collected, accounting for 0.4% (10/2547) of all E. coli obtained from urine samples, 0.9% (10/1135) of ampicillin-susceptible E. coli isolates and 9.6% (10/104) of MEC-R E. coli isolates. The isolates appeared as small colonies with round morphology and had impaired fitness. The isolates were not clonal and belonged to various extraintestinal or commensal E. coli phylogroups. Mutations in the cysB gene were evidenced in all clinical isolates. In conclusion, microbiologists should be aware of these isolates with a particular susceptibility phenotype, which is not due to error in disk diffusion but is a real non-enzymatic antibiotic resistance pattern.

Occupational Exposures to Ebola Virus in Ebola Treatment Center, Conakry, Guinea.

We report 77 cases of occupational exposures for 57 healthcare workers at the Ebola Treatment Center in Conakry, Guinea, during the Ebola virus disease outbreak in 2014-2015. Despite the high incidence of 3.5 occupational exposures/healthcare worker/year, only 18% of workers were at high risk for transmission, and no infections occurred.

Decontamination of a field laboratory dedicated to Ebola virus-infected patients.

In 2015, the French Armed Forces deployed a biosafety level 3 (BSL3) field laboratory as a part of an Ebola treatment center in Guinea. When closing the center, laboratory decontamination operations were necessary. We present the decontamination protocols applied for the BSL3 field laboratory, making the entire module ready for a future use.

Ebola Virus RNA Stability in Human Blood and Urine in West Africa's Environmental Conditions.

We evaluated RNA stability of Ebola virus in EDTA blood and urine samples collected from infected patients and stored in West Africa's environmental conditions. In blood, RNA was stable for at least 18 days when initial cycle threshold values were <30, but in urine, RNA degradation occurred more quickly.

Galleria mellonella as an infection model for select agents.

The use of animal models is a key step to better understand bacterial virulence factors and their roles in host/pathogen interactions. To avoid the ethical and cost problems of mammalian models in bacterial virulence research, several insect models have been developed. One of these models, the larvae of the greater wax moth Galleria mellonella, has been shown to be relevant for several fungal and bacterial mammalian pathogens. Here, we describe the use G. mellonella to study virulence of the highly virulent facultative intracellular bacterial pathogens: Brucella suis, Brucella melitensis, Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei.

The condition-dependent transcriptional landscape of Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes--Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes--quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an "accidental pathogen", where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts.

Interplay between three RND efflux pumps in doxycycline-selected strains of Burkholderia thailandensis.

BACKGROUND: Efflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure. METHODOLOGY/PRINCIPAL FINDINGS: We used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect. CONCLUSIONS/SIGNIFICANCE: These results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors.

Involvement of the efflux pumps in chloramphenicol selected strains of Burkholderia thailandensis: proteomic and mechanistic evidence.

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some ß-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.

Phenotypic and genetic characterization of macrolide resistance in Francisella tularensis subsp. holarctica biovar I.

OBJECTIVES: Francisella tularensis subsp. holarctica strains are classified as biovars I and II, which are susceptible and naturally resistant to the macrolide erythromycin, respectively. The present study was aimed at both selecting biovar I strains with increased levels of erythromycin resistance and characterizing the underlying genetic mechanisms. METHODS: Serial cultures in the presence of increasingly high erythromycin concentrations were performed to select independent high- and intermediate-level erythromycin-resistant mutants from each of three different biovar I strains. The mutants were characterized for cross-resistance to several antibiotics, presence of mutations in the genes encoding the 23S rRNA and the L4 and L22 ribosomal proteins, and overexpression of efflux pumps. RESULTS: Mutants displayed cross-resistance to all macrolide compounds tested but not to other classes of antibiotics. We found mutations in domain V of the 23S rRNA gene (G2057A, A2058G, A2058T and C2611T) and in the gene encoding L22, leading to either the G91D substitution or the M82K83R84 deletion. Analysis of mutants with intermediate resistance levels obtained over the course of the selection process revealed both a positive correlation between the number of mutated ribosomal operons and the resistance level, and an additional resistance mechanism in the early steps of selection. CONCLUSIONS: We showed that high-level resistance to macrolides can be easily obtained in vitro in F. tularensis subsp. holarctica biovar I strains, thereby suggesting that in vivo selection for resistance may explain reported failures of antibiotic treatment. Ketolides were the most effective macrolides tested, which may limit the risk of selection for resistance.