Description: The control of multihost pathogens, such as Coxiella burnetii , should rely on accurate information about the roles played by the main hosts. We aimed to determine the involvement of the red deer ( Cervus elaphus ) in the ecology of C. burnetii . We predicted that red deer populations from broad geographic areas within a European context would be exposed to C. burnetii , and therefore, we hypothesized that a series of factors would modulate the exposure of red deer to C. burnetii . To test this hypothesis, we designed a retrospective survey of 47 Iberian red deer populations from which 1,751 serum samples and 489 spleen samples were collected. Sera were analyzed by enzyme-linked immunosorbent assays (ELISA) in order to estimate exposure to C. burnetii , and spleen samples were analyzed by PCR in order to estimate the prevalence of systemic infections. Thereafter, we gathered 23 variables—within environmental, host, and management factors—potentially modulating the risk of exposure of deer to C. burnetii , and we performed multivariate statistical analyses to identify the main risk factors. Twenty-three populations were seropositive (48.9%), and C. burnetii DNA in the spleen was detected in 50% of the populations analyzed. The statistical analyses reflect the complexity of C. burnetii ecology and suggest that although red deer may maintain the circulation of C. burnetii without third species, the most frequent scenario probably includes other wild and domestic host species. These findings, taken together with previous evidence of C. burnetii shedding by naturally infected red deer, point at this wild ungulate as a true reservoir for C. burnetii and an important node in the life cycle of C. burnetii , at least in the Iberian Peninsula.

Description: Streptococcus pneumoniae (pneumococcus) is both a widespread nasal colonizer and a leading cause of otitis media, one of the most common diseases of childhood. Pneumococcal phase variation influences both colonization and disease and thus has been linked to the bacteria's transition from colonizer to otopathogen. Further contributing to this transition, coinfection with influenza A virus has been strongly associated epidemiologically with the dissemination of pneumococci from the nasopharynx to the middle ear. Using a mouse infection model, we demonstrated that coinfection with influenza virus and pneumococci enhanced both colonization and inflammatory responses within the nasopharynx and middle ear chamber. Coinfection studies were also performed using pneumococcal populations enriched for opaque or transparent phase variants. As shown previously, opaque variants were less able to colonize the nasopharynx. In vitro , this phase also demonstrated diminished biofilm viability and epithelial adherence. However, coinfection with influenza virus ameliorated this colonization defect in vivo . Further, viral coinfection ultimately induced a similar magnitude of middle ear infection by both phase variants. These data indicate that despite inherent differences in colonization, the influenza A virus exacerbation of experimental middle ear infection is independent of the pneumococcal phase. These findings provide new insights into the synergistic link between pneumococcus and influenza virus in the context of otitis media.

Description: Nontypeable Haemophilus influenzae (NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded by hktE ) and a bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX ) in resistance of NTHI to oxidants and persistence in vivo . Isogenic NTHI strain 86-028NP mutants lacking hktE and pdgX had increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed that pdgX and hktE were important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilms in vivo . Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.

Description: The presence of Bartonella spp. was investigated in domestic ungulates grazing in communal pastures from a mountain area in northern Spain, where 18.3% (17/93) of cattle were found to be positive by PCR combined with a reverse line blot (PCR/RLB), whereas sheep ( n = 133) or horses ( n = 91) were found not to be infected by this pathogen. Bartonella infection was significantly associated with age, since older animals showed a higher prevalence than heifers and calves. In contrast to other studies, B. chomelii was the most frequent species found in cattle (14/17), while B. bovis was detected in only three animals. Moreover, 18 B. chomelii isolates and one B. bovis isolate were obtained from nine animals. Afterwards, B. chomelii isolates were characterized by a multilocus sequence typing (MLST) method which was adapted in this study. This method presented a high discrimination power, identifying nine different sequence types (STs). This characterization also showed the presence of different STs simultaneously in the same host and that STs had switched over time in one of the animals. In addition, B. chomelii STs seem to group phylogenetically in two different lineages. The only B. bovis isolate was characterized with a previously described MLST method. This isolate corresponded to a new ST which is located in lineage I, where the B. bovis strains infecting Bos taurus subsp. taurus are grouped. Further studies on the dynamics of Bartonella infection in cattle and the potential ectoparasites involved in the transmission of this microorganism should be performed, improving knowledge about the interaction of Bartonella spp. and domestic ungulates.

Description: We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum β-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC ( T 〉MIC ) below clinically achievable levels in humans. These findings provide evidence supporting the potential of β-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Description: Tigecycline is a translational inhibitor with efficacy against a wide range of pathogens. Using experimental evolution, we adapted Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Staphylococcus aureus to growth in elevated tigecycline concentrations. At the end of adaptation, 35 out of 47 replicate populations had clones with a mutation in rpsJ , the gene that encodes the ribosomal S10 protein. To validate the role of mutations in rpsJ in conferring tigecycline resistance, we showed that mutation of rpsJ alone in Enterococcus faecalis was sufficient to increase the tigecycline MIC to the clinical breakpoint of 0.5 μg/ml. Importantly, we also report the first identification of rpsJ mutations associated with decreased tigecycline susceptibility in A. baumannii , E. coli , and S. aureus . The identified S10 mutations across both Gram-positive and -negative species cluster in the vertex of an extended loop that is located near the tigecycline-binding pocket within the 16S rRNA. These data indicate that S10 is a general target of tigecycline adaptation and a relevant marker for detecting reduced susceptibility in both Gram-positive and -negative pathogens.

Description: Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn 2+ -inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn 2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene ( smtA 7942 ) and its cognate SmtB 7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin ( cpcBA 6803 ) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster β-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. IMPORTANCE This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn 2+ -inducible system, which is based on the smtA 7942 operator/promoter and smtB 7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

Description: Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae . The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae . However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli , the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae , EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB , thus conferring antimicrobial resistance as well as a stress response in E. cloacae . In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae .