Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.

Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7RB is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC712 deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7RB showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7RB were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7RB was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to ß-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.

A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

In vivo immunogenicity assessment and vaccine efficacy evaluation of a chimeric tandem repeat of epitopic region of OMP31 antigen fused to interleukin 2 (IL-2) against Brucella melitensis in BALB/c mice.

BACKGROUND: Designing a potent recombinant vaccine, using the appropriate subunits with the greatest effect on stimulating the immune system, especially in the case of intracellular pathogens such as gram negative Brucella Melitensis bacteria, is of great importance. In this study, three repeats of 27 amino acids of the immunogenic epitope derived from OMP31 antigen (3E) from the Brucella melitensis, in a protective manner against Brucellosis have been used. To fortify the delivery system of recombinant antigens, IL-2 cytokine as a molecular adjuvant was fused to recombinant constructs. Recombinant proteins were evaluated for immunological studies in a mouse model (BALB/c). RESULTS: The results showed that all recombinant proteins could stimulate the immune system to produce Th1 cytokines and antibodies in compare to the negative control treatments. 3E-IL2 and then OMP31-IL2 proteins stimulated higher levels of IFN-γ and IL-2 compared to the other treatments (p < 0.05). Also, the results indicated that experimental treatments produced a higher level of IgG2a isotype than IgG1 isotype. In addition, the findings of the experiment showed that the presence of chemical adjuvant (IFA) along with molecular adjuvant can play a significant role in stimulating the immune system. After determining the potency of recombinant structures, their efficacy in stimulating the immune system were also evaluated. B. melitensis M16 strain was used to challenge 30 days after last immunization. The microbial load of the splenocyte in the treatments receiving chimeric proteins were significantly lower. Also, Wright serological test confirmed that these treatments had the lowest agglutination rate, as well as the positive treatment, while in the negative treatments in excess of blood serum dilutions, agglutination rate were more than 2 + . CONCLUSIONS: 3E-IL2 treatment showed the best performance compared to other recombinant proteins and could be considered as the suitable candidate for further research on the production of recombinant vaccine against Brucella.

Cloning and Expression of Mannheimia haemolytica PlpE Gene in Escherichia coli and its Immunogenicity Assessment.

Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-&beta;-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.

Molecular Assessment of Anaplasma marginale in Bovine and Rhipicephalus (Boophilus) microplus Tick of Endemic Tribal Belt of Coastal South Gujarat, India.

BACKGROUND AND METHODS: This study described the detection, prevalence and phylogeny of Anaplasma marginale in the bovine (cattle and buffaloes) and Rhipicephalus (Boophilus) microplus tick belonged to the tribal area of coastal South Gujarat, India, by amplifying 576 bp of major surface protein (msp) 5 gene using custom designed primers in the polymerase chain reaction (PCR). RESULTS: The PCR detection limit was up to 20 parasites/µl of blood in sensitivity experiment, and observed 100% specificity against Trypanosoma evansi, Babesia bigemina and Theileria annulata. Prevalence rate of the A. marginale in the bovine (n = 211)) was 18.48% and 6.64% (p < 0.05) as per the PCR and Giemsa stained blood smear, respectively. Febrile animals (35%) observed significantly (p < 0.05) higher incidence rate than the non-febrile (14.62%). The amplified msp5 had single cut site for the EcoR1 enzyme, upon digestion yielded two fragments of 365 and 211 bp on 1.0% agarose gel. The current sequence (KC811329) showed 100% homology and 1064 total score with the published nucleotide sequences of msp5 of A. marginale in the NCBI-BLAST study. Monophyletic relationship was observed with high bootstrap proportion (> 76% in Neighbor-Joining/ Maximum Likelihood) between the current and published nucleotide sequences in the phylogeny. Twenty out of 39 A. marginale infected bovine recorded R. (B.) microplus on their body surface, out of which 18 had detected the infection. The rickettsia was in 55%, 65% and 25% of anterior half, posterior half and egg of tick, respectively. CONCLUSION: The test detected A. marginale in a carrier, pre-symptomatic and symptomatic vertebrate hosts (cattle and buffalo) and different body parts of the starved R. (B.) microplus including its egg. The current genotype could be an explanation for the frequent outbreaks of bovine anaplasmosis in the targeted areas.

Fitness cost of a mcr-1-carrying IncHI2 plasmid.

IncHI2 is a common type of large mcr-1-carrying plasmids that have been found worldwide. Large plasmids could impose metabolic burden for host bacterial strains, we therefore examine the stability and fitness cost of a mcr-1-carrying 265.5-kb IncHI2 plasmid, pMCR1_1943, in Escherichia coli in nutrient-rich LB and nutrient-restricted M9 broth. Stability tests revealed that pMCR1_1943 was stably maintained with a stability frequency of 0.99±0.01 (mean ± standard deviation) after 880 generations in LB and 0.97±0.00 after 220 generations in M9 broth. Relative fitness (expressed as w, defined as relative fitness of the plasmid-carrying strain compared to the plasmid-free progenitor strain) was examined using the 24-h head to head competitions. pMCR1_1943 initially imposed costs (w, 0.88±0.03 in LB, 0.87±0.01 in M9) but such costs were largely reduced after 14-day cultures (w, 0.97±0.03 in LB, 0.95±0.03 in M9). The stable maintenance and the largely compensated cost after passage may contribute to the wide spread of mcr-1-carrying IncHI2 plasmids. To investigate potential mechanisms for the reduced fitness cost, we performed whole genome sequencing and single nucleotide polymorphism calling for the competitor strains. We identified that molecular chaperone-encoding dnaK, cell division protein-encoding cpoB and repeat protein-encoding rhsC were associated with the cost reduction for pMCR1_1943, which may represent new mechanisms for host bacterial strains to compensate fitness costs imposed by large plasmids and warrant further studies.

Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps.

It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H+ accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations.IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not always outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensating for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the development of drugs that will reduce the persistence of resistance by increasing said costs.

Ligand-Mediated Folding of the OmpA Periplasmic Domain from Acinetobacter baumannii.

The periplasmic domain of OmpA from Acinetobacter baumannii (AbOmpA-PD) binds to diaminopimelate and anchors the outer membrane to the peptidoglycan layer in the cell wall. Although the crystal structure of AbOmpA-PD with its ligands has been reported, the mechanism of ligand-mediated folding of AbOmpA remains elusive. Here, we report that in vitro refolded apo-AbOmpA-PD in the absence of ligand exists as a mixture of two partially folded forms in solution: mostly unfolded (apo-state I) and hololike (apo-state II) states. Binding of the diaminopimelate or glycine ligand induced complete folding of AbOmpA-PD. The apo-state I was highly flexible and contained some secondary structural elements, whereas the apo-state II closely resembled the holo-state in terms of both structure and backbone dynamics, except for the ligand-binding region. 15N-relaxation-dispersion analyses for apo-state II revealed substantial motion on a millisecond timescale of residues in the H3 helix near the ligand-binding site, with this motion disappearing upon ligand binding. These results provide an insight into the ligand-mediated folding mechanism of AbOmpA-PD in solution.

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.

Proteomic Assessment of the Expression of Genes Related to Toluene Catabolism and Porin Synthesis in Pseudomonas stutzeri ST-9.

The organization and expression of Pseudomonas stutzeri ST-9 genes related to toluene catabolism and porin synthesis was investigated. Toluene-degrading genes were found to be localized in the chromosome close to a phage-type integrase. A regulatory gene and 21 genes related to an aromatics degradation pathway are organized as a putative operon. These proteins are upregulated in the presence of toluene. Fourteen outer membrane proteins were identified as porins in the ST-9 genome. The identified porins showed that the main detected porins are related to the OmpA and OprD superfamilies. The percentage of porins in the outer membrane protein fraction, as determined by mass spectrometry, was 73% and 54% when the cells were cultured with toluene and with glucose, respectively. Upregulation of OmpA and downregulation of OprD occurred in the presence of toluene. A porin fraction (90% OprD) from both cultures was isolated and examined as a toluene uptake system using the liposome-swelling assay. Liposomes were prepared with the porin fraction from a culture that was grown on toluene (T-proteoliposome) or glucose (G-proteoliposome). There was no significant difference in the permeability rate of the different solutes through the T-proteoliposome and the G-proteoliposome.

Amelioration of the Fitness Costs of Antibiotic Resistance Due To Reduced Outer Membrane Permeability by Upregulation of Alternative Porins.

The fitness cost of antibiotic resistance is a key parameter in determining the evolutionary success of resistant bacteria. Studies of the effect of antibiotic resistance on bacterial fitness are heavily biased toward target alterations. Here we investigated how the costs in the form of a severely impaired growth rate associated with resistance due to absence of two major outer membrane porins can be genetically compensated. We performed an evolution experiment with 16 lineages of a double mutant of Escherichia coli with the ompCF genes deleted, and reduced fitness and increased resistance to different classes of antibiotics, including the carbapenems ertapenem and meropenem. After serial passage for only 250 generations, the relative growth rate increased from 0.85 to 0.99 (susceptible wild type set to 1.0). Compensation of the costs followed two different adaptive pathways where upregulation of expression of alternative porins bypassed the need for functional OmpCF porins. The first compensatory mechanism involved mutations in the phoR and pstS genes, causing constitutive high-level expression of the PhoE porin. The second mechanism involved mutations in the hfq and chiX genes that disrupted Hfq-dependent small RNA regulation, causing overexpression of the ChiP porin. Although susceptibility was restored in compensated mutants with PhoE overexpression, evolved mutants with high ChiP expression maintained the resistance phenotype. Our findings may explain why porin composition is often altered in resistant clinical isolates and provide new insights into how bypass mechanisms may allow genetic adaptation to a common multidrug resistance mechanism.

Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

Evaluation of the Antibiotic Resistance and Virulence of Escherichia coli Strains Isolated from Chicken Carcasses in 2007 and 2013 from Paraná, Brazil.

The frequent use of antimicrobials in commercial poultry production has raised concerns regarding the potential impact of antimicrobials on human health due to selection for resistant bacteria. Several studies have reported similarities between extraintestinal pathogenic Escherichia coli (ExPEC) strains isolated from birds and humans, indicating that these contaminant bacteria in poultry may be linked to human disease. The aim of our study was to analyze the frequency of antimicrobial resistance and virulence factors among E. coli strains isolated from commercial chicken carcasses in Paraná, Brazil, in 2007 and 2013. A total of 84 E. coli strains were isolated from chicken carcasses in 2007, and 121 E. coli strains were isolated in 2013. Polymerase chain reaction was used to detect virulence genes (hlyF, iss, ompT, iron, and iutA) and to determine phylogenetic classification. Antimicrobial susceptibility testing was performed using 15 antimicrobials. The strains were also confirmed as extended-spectrum ß-lactamase (ESBL)-producing E. coli with phenotypic and genotypic tests. The results indicated that our strains harbored virulence genes characteristic of ExPEC, with the iutA gene being the most prevalent. The phylogenetic groups D and B1 were the most prevalent among the strains isolated in 2007 and 2013, respectively. There was an increase in the frequency of resistance to a majority of antimicrobials tested. An important finding in this study was the large number of ESBL-producing E. coli strains isolated from chicken carcasses in 2013, primarily for the group 2 cefotaximase (CTX-M) enzyme. ESBL production confers broad-spectrum resistance and is a health risk because ESBL genes are transferable from food-producing animals to humans via poultry meat. These findings suggest that our strains harbored virulence and resistance genes, which are often associated with plasmids that can facilitate their transmission between bacteria derived from different hosts, suggesting zoonotic risks.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle.

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.

Metabolic compensation of fitness costs associated with overexpression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa.

The acquisition of antibiotic resistance has been associated with a possible nonspecific, metabolic burden that is reflected in decreased fitness among resistant bacteria. We have recently demonstrated that overexpression of the MexEF-OprN multidrug efflux pump does not produce a metabolic burden when measured by classical competitions tests but rather leads to a number of changes in the organism's physiology. One of these changes is the untimely activation of the nitrate respiratory chain under aerobic conditions. MexEF-OprN is a proton/substrate antiporter. Overexpression of this element should result in a constant influx of protons, which may lead to cytoplasmic acidification. Acidification was not observed in aerobiosis, a situation in which the MexEF-overproducing mutant increases oxygen consumption. This enhanced oxygen uptake serves to eliminate intracellular proton accumulation, preventing the cytoplasmic acidification that was observed exclusively under anaerobic conditions, a situation in which the fitness of the MexEF-OprN-overproducing mutant decreases. Finally, we determined that the early activation of the nitrate respiratory chain under aerobic conditions plays a role in preventing a deleterious effect associated with the overexpression of MexEF-OprN. Our results show that metabolic rewiring may assist in overcoming the potential fitness cost associated with the acquisition of antibiotic resistance. Furthermore, the capability to metabolically compensate for this effect is habitat dependent, as demonstrated by our results under anaerobic conditions. The development of drugs that prevent metabolic compensation of fitness costs may help to reduce the persistence and dissemination of antibiotic resistance.

Assessment of conjugal transfer of antibiotic resistance genes in Salmonella Typhimurium exposed to bile salts.

This study was designed to evaluate the transfer potential of antibiotic resistance genes in antibiotic-resistant Salmonella Typhimurium (S. Typhimurium(R)) in the presence of bile salts. The resistance of S. Typhimurium(R) to ampicillin, kanamycin, and tetracycline was increased by 64-, 64-, and 512-fold, respectively. The highest transfer frequency from S. Typhimurium(R) to Escherichia coli was observed at the bile salt concentration of 160 µg/ml (3.8 × 10(-3) transferrants/cells). The expression of traJ and traY was suppressed in S. Typhimurium(R) by bile salt. This study provides useful information for understanding the conjugative transfer of antibiotic resistance genes in S. Typhimurium under intestinal conditions.

Genetic assessment of the role of AcrB ß-hairpins in the assembly of the TolC-AcrAB multidrug efflux pump of Escherichia coli.

The tripartite AcrAB-TolC multidrug efflux pump of Escherichia coli is the central conduit for cell-toxic compounds and contributes to antibiotic resistance. While high-resolution structures of all three proteins have been solved, much remains to be learned as to how the individual components come together to form a functional complex. In this study, we investigated the importance of the AcrB ß-hairpins belonging to the DN and DC subdomains, which are presumed to dock with TolC, in complex stability and activity of the complete pump. Our data show that the DN subdomain ß-hairpin residues play a more critical role in complex stability and activity than the DC subdomain hairpin residues. The failure of the AcrB DN ß-hairpin deletion mutant to engage with TolC leads to the drug hypersensitivity phenotype, which is reversed by compensatory alterations in the lipoyl and ß-barrel domains of AcrA. Moreover, AcrA and TolC mutants that induce TolC opening also reverse the drug hypersensitivity phenotype of the AcrB ß-hairpin mutants, indicating a failure by the AcrB mutant to interact and thus induce TolC opening on its own. Together, these data suggest that both AcrB ß-hairpins and AcrA act to stabilize the tripartite complex and induce TolC opening for drug expulsion.

Assessment of the potential contribution of the highly conserved C-terminal motif (C10) of Borrelia burgdorferi outer surface protein C in transmission and infectivity.

OspC is produced by all species of the Borrelia burgdorferi sensu lato complex and is required for infectivity in mammals. To test the hypothesis that the conserved C-terminal motif (C10) of OspC is required for function in vivo, a mutant B. burgdorferi strain (B31::ospCΔC10) was created in which ospC was replaced with an ospC gene lacking the C10 motif. The ability of the mutant to infect mice was investigated using tick transmission and needle inoculation. Infectivity was assessed by cultivation, qRT-PCR, and measurement of IgG antibody responses. B31::ospCΔC10 retained the ability to infect mice by both needle and tick challenge and was competent to survive in ticks after exposure to the blood meal. To determine whether recombinant OspC protein lacking the C-terminal 10 amino acid residues (rOspCΔC10) can bind plasminogen, the only known mammalian-derived ligand for OspC, binding analyses were performed. Deletion of the C10 motif resulted in a statistically significant decrease in plasminogen binding. Although deletion of the C10 motif influenced plasminogen binding, it can be concluded that the C10 motif is not required for OspC to carry out its critical in vivo functions in tick to mouse transmission.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.