Tigecycline Sensitivity Reduction in Escherichia coli Due to Widely Distributed tet(A) Variants.

Despite scattered studies that have reported mutations in the tet(A) gene potentially linked to tigecycline resistance in clinical pathogens, the detailed function and epidemiology of these tet(A) variants remains limited. In this study, we analyzed 64 Escherichia coli isolates derived from MacConkey plates supplemented with tigecycline (2 µg/mL) and identified five distinct tet(A) variants that account for reduced sensitivity to tigecycline. In contrast to varied tigecycline MICs (0.25 to 16 µg/mL) of the 64 tet(A)-variant-positive E. coli isolates, gene function analysis confirmed that the five tet(A) variants exhibited a similar capacity to reduce tigecycline sensitivity in DH5α carrying pUC19. Among the observed seven non-synonymous mutations, the V55M mutation was unequivocally validated for its positive role in conferring tigecycline resistance. Interestingly, the variability in tigecycline MICs among the E. coli strains did not correlate with tet(A) gene expression. Instead, a statistically significant reduction in intracellular tigecycline concentrations was noted in strains displaying higher MICs. Genomic analysis of 30 representative E. coli isolates revealed that tet(A) variants predominantly resided on plasmids (n = 14) and circular intermediates (n = 13). Within China, analysis of a well-characterized E. coli collection isolated from pigs and chickens in 2018 revealed the presence of eight tet(A) variants in 103 (4.2%, 95% CI: 3.4-5.0%) isolates across 13 out of 17 tested Chinese provinces or municipalities. Globally, BLASTN analysis identified 21 tet(A) variants in approximately 20.19% (49,423/244,764) of E. coli genomes in the Pathogen Detection database. These mutant tet(A) genes have been widely disseminated among E. coli isolates from humans, food animals, and the environment sectors, exhibiting a growing trend in tet(A) variants over five decades. Our findings underscore the urgency of addressing tigecycline resistance and the underestimated role of tet(A) mutations in this context.

Molecular Surveillance of MRSA in Raw Milk Provides Insight into MRSA Cross Species Evolution.

Methicillin-resistant Staphylococcus aureus (MRSA) in foods has been associated with severe infections in humans and animals worldwide. In the present study, the molecular characteristics of livestock-associated MRSA (LA-MRSA) and human-associated MRSA (hMRSA) isolates obtained in China, as well as MRSA isolates obtained from raw milk in 2018, were investigated. In total, 343 (20.38%; 343/1,683) S. aureus isolates were obtained from 1,683 raw milk samples from 100 dairy farms in 11 provinces across China. Among these, 49 (2.91%; 49/1,683) were mecA-positive MRSA. All LA-MRSA isolates were resistant to penicillin and highly resistant to erythromycin, sulfisoxazole, and clindamycin. Bioinformatic analysis the 49 genomes of LA-MRSA and 71 previously published hMRSA genomes isolated from Chinese individuals in 2018 indicated that blaZ, erm, ant(6)-Ia, aph(3')-III, tet(K), cat, and aph(2″)-Ia were more prevalent in MRSA from raw milk (P < 0.05) compared to hMRSA. Additionally, hMRSA isolates were more significantly associated with ST5 (P < 0.01) compared to LA-MRSA; in contrast, ST338 was more prevalent among LA-MRSA isolates (P < 0.01). Likewise, the SCCmec type II was only detected in hMRSA isolates, whereas SCCmec type V and IV were more prevalent among LA-MRSA (P < 0.01). Furthermore, core-genome phylogenetic analysis showed the endemic characteristics of LA-MRSA in local provinces, as well as the close evolutionary relationships between MRSA from cattle and humans. Finally, homology analysis of mecA and blaZ genetic contexts revealed a high possibility of horizontal transmission of MRSA resistance genes among raw milk-associated and hMRSA strains, which increases the risk for public health. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is considered a public health concern as it is resistant to multiple antibiotics, thus being in zoonotic transmission of antibiotic resistance genes. MRSA causes serious public health issues and leads to hard-to-treat infections in humans and animals; therefore, it was meaningful to determine the prevalence of MRSA in raw milk samples and investigate phenotype and genotype of antimicrobial resistance and molecular characteristics in livestock-associated MRSA (LA-MRSA) and human-associated MRSA (hMRSA) in China, which could provide a theoretical basis for preventing and controlling the spread of MRSA between livestock and humans.

Development of a Real-Time Recombinase-Aided Amplification Method to Rapidly Detect Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant staphylococcus aureus (MRSA) is a major pathogen responsible for human hospital and community-onset diseases and severe invasive livestock infections. Rapid detection of MRSA is essential to control the spread of MRSA. Conventional identification methods and antibacterial susceptibility tests of MRSA are time-consuming. The commonly used qPCR assay also has the disadvantages of being complicated and expensive, restricting its application in resource-limited clinical laboratories. Here, a real-time fluorescent recombinase-assisted amplification (RAA) assay targeting the most conserved regions within the mecA gene of MRSA was developed and evaluated to detect MRSA. The detection limit of this assay was determined to be 10 copies/reaction of positive plasmids. The established RAA assay showed high specificity for MRSA detection without cross-reactivities with other clinically relevant bacteria. The diagnostic performance of real-time RAA was evaluated using 67 clinical S. aureus isolates from dairy farms, which were detected in parallel using the TaqMan probe qPCR assay. The results showed that 56 and 54 samples tested positive for MRSA by RAA and qPCR, respectively. The overall agreement between both assays was 97.01% (65/67), with a kappa value of 0.9517 (p < 0.001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R2 = 0.9012, p < 0.0001), indicating that this RAA assay possesses similar detection performance to the qPCR assay. In conclusion, our newly established RAA assay is a time-saving and convenient diagnostic tool suitable for MRSA detection and screening.

New clues about the global MRSA ST398: Emergence of MRSA ST398 from pigs in Qinghai, China.

This study, a part of the China national surveillance program on antimicrobial resistance in zoonotic bacteria, was to determine the phenotypic and genomic characteristics of endemic pig-associated Staphylococcus aureus ST398 strains in China. A total of 68 (48.9 %) S. aureus strains were recovered from 139 samples collected from two pig farms and one slaughterhouse in Qinghai province. Genomic characterization of All S. aureus strains was performed by WGS and their evolutionary relationships were assessed by phylogenetic analysis. Their susceptibilities to antimicrobials were determined using the broth dilution method. All S. aureus strains consisted of 41 ST398-t571 MSSA, 26 ST398-t011 MRSA and 1 ST5-t002 MRSA. Among these, ST398 was frequently identified in 67 S. aureus strains, suggesting that ST398 was a frequent source of MRSA and MSSA infections in Qinghai province and its possibility of transmission between individuals in pigs from farms and slaughterhouse. Meanwhile, Livestock-associated-MRSA ST398 in our study was establishing closely evolutionary relationships with MRSA ST 398 in Europe and Australia. The clues about closely relatedness of the global S. aureus ST398 underscore the potential public health risk of S. aureus ST398 in the pork supply chain and offer significant guidance for veterinary and human health.

Macrophage membrane modified baicalin liposomes improve brain targeting for alleviating cerebral ischemia reperfusion injury.

Baicalin (BA) has a good intervention effect on encephalopathy. In this study, macrophage membrane was modified on the surface of baicalin liposomes (BA-LP) by extrusion method. Macrophage membrane modified BA-LP (MM-BA-LP) was characterized by various analytical techniques, and evaluated for brain targeting. The results presented MM-BA-LP had better brain targeting compared with BA-LP. Pharmacokinetic experiments showed that MM-BA-LP improved pharmacokinetic parameters and increased the residence time of BA. Pharmacodynamic of middle cerebral artery occlusion (MCAO) rat model was studied to verify the therapeutic effect of MM-BA-LP on cerebral ischemia reperfusion injury (CIRI). The results showed that MM-BA-LP could significantly improve the neurological deficit, cerebral infarction volume and brain pathological state of MCAO rats compared with BA-LP. These results suggested that MM-BA-LP could significantly enhance the brain targeting and improve the circulation of BA in blood, and had a significantly better neuroprotective effect on MCAO rats than BA-LP.

Prevalence of Salmonella and Antimicrobial Resistance in Isolates from Food Animals - Six PLADs, China, 2019.

What is already known about this topic? Salmonella causes acute and chronic diseases in food animals, and infected food animals are one of the most important source of human infection. What does this report contribute? The prevalence of Salmonella was 10.5% in chicken samples, 24.4% in pig, 23.3% in duck, and 29.4% in milk. Salmonella isolates were highly resistant to ampicillin (59.60%). What are the implications for public health practices? Data on Salmonella infections among food animals in China could help identify sources and factors related to the spread of Salmonella in food animals and food production chains.

Borneol in cardio-cerebrovascular diseases: Pharmacological actions, mechanisms, and therapeutics.

With the coming acceleration of global population aging, the incidence rate of cardio-cerebrovascular diseases (CVDs) is increasing. It has become the leading cause of human mortality. As a natural drug, borneol (BO) not only has anti-inflammatory, anti-oxidant, anti-apoptotic, anti-coagulant activities and improves energy metabolism but can also promote drugs to enter the target organs or tissues through various physiological barriers, such as the blood-brain barrier (BBB), mucous membrane, skin. Thus, it has a significant therapeutic effect on various CVDs, which has been confirmed in a large number of studies. However, the pharmacological actions and mechanisms of BO on CVDs have not been fully investigated. Hence, this review summarizes the pharmacological actions and possible mechanisms of BO, which provides novel ideas for the treatment of CVDs.

Baicalin Liposome Alleviates Lipopolysaccharide-Induced Acute Lung Injury in Mice via Inhibiting TLR4/JNK/ERK/NF-κB Pathway.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are challenging diseases with the high mortality in a clinical setting. Baicalin (BA) is the main effective constituent isolated from the Chinese medical herb Scutellaria baicalensis Georgi, and studies have proved that it has a protective effect on ALI induced by lipopolysaccharide (LPS) due to the anti-inflammatory efficacy. However, BA has low solubility which may limit its clinical application. Hence, we prepared a novel drug delivery system-Baicalin liposome (BA-LP) in previous research-which can improve some physical properties of BA. Therefore, we aimed to explore the effect of BA-LP on ALI mice induced by LPS. In pharmacokinetics study, the values of t 1/2 and AUC0- t in the BA-LP group were significantly higher than that of the BA group in normal mice, indicating that BA-LP could prolong the duration time in vivo of BA. The BA-LP group also showed a higher concentration in lung tissues than the BA group. Pharmacodynamics studies showed that BA-LP had a better effect than the BA group at the same dosage on reducing the W/D ratio, alleviating the lung injury score, and decreasing the proinflammatory factors (TNF-α, IL-1ß) and total proteins in bronchoalveolar lavage fluids (BALF). In addition, the therapeutic effects of BA-LP showed a dose-dependent manner. Western blot analysis indicated that the anti-inflammatory action of BA could be attributed to the inhibition of the TLR4-NFκBp65 and JNK-ERK signaling pathways. These results suggest that BA-LP could be a valuable therapeutic candidate in the treatment of ALI.

Preparation, Characterization and in vivo Study of Borneol-Baicalin-Liposomes for Treatment of Cerebral Ischemia-Reperfusion Injury.

PURPOSE: Baicalin (BA) has a good neuroprotective effect, but it is eliminated quickly in the body and does not easily reach the brain. In this experiment, borneol (BO) was used as an auxiliary drug to prepare borneol-baicalin-liposomes (BO-BA-LP) to prolong the efficacy time of BA, synergistically synergize, introduce drugs into the brain, and better exert the therapeutic effect on cerebral ischemia-reperfusion (I/R) injury. METHODS: Through single-factor inspection and response surface optimization analysis, obtained the best preparation process of BO-BA-LP and characterized by various analytical techniques. Validated the long-term effectiveness of BA-BO-LP through pharmacokinetic studies and conducted pharmacodynamic studies on the middle cerebral artery occlusion (MCAO) rat model to verify the therapeutic effect of BO-BA-LP on cerebral I/R injury. RESULTS: The optimum preparation conditions of BO-BA-LP were as follows: the dosage of BO was 9.55 mg, the ratio of phospholipid to drug was 4.02:1, the ratio of phospholipid to cholesterol was 7.25:1, the entrapment efficiency (EE) was 41.49%, and the drug loading (DL) was 4.29%. The particle size range of the liposomes was 167.1 nm, and the polydispersity index (PDI) range was 0.113. The results of pharmacokinetic experiments showed that the combination of BA and BO liposomes effectively improved the pharmacokinetic parameters of BA and prolonged the half-life of BA. Pharmacodynamic studies have found that, compared with BA-LP, BO-BA-LP can significantly improve neurological deficits, cerebral infarction volume, and brain pathological states on MCAO rats. CONCLUSION: These results demonstrated that BO-BA-LP can improve the circulation of drugs in the blood, and the addition of BO can enhance the therapeutic effect of BA and effectively improve cerebral I/R.

Emergence of livestock-associated MRSA ST398 from bulk tank milk, China.

OBJECTIVES: To detect livestock-associated MRSA (LA-MRSA) ST398 from bulk tank milk in China and to determine the phenotypic and genomic characteristics of the strains. METHODS: LA-MRSA ST398 strains were isolated from bulk tank milk samples in Shanghai and their susceptibilities to antimicrobials were determined using the broth dilution method. Genomic characterization of MRSA ST398 strains was performed by WGS and their evolutionary relationships were assessed by phylogenetic analysis. RESULTS: Two LA-MRSA ST398 isolates were recovered from bulk tank milk samples in two geographically distant farms in China. Whole-genome analysis strongly suggested that the LA-MRSA ST398 strains were closely related to the highly virulent hospital-associated MRSA (HA-MRSA) ST398 strains in China. CONCLUSIONS: The presence of LA-MRSA ST398 in bulk tank milk might be a serious threat to public health, highlighting the need for active surveillance of LA-MRSA in healthy cattle in China.

Genomic epidemiology of animal-derived tigecycline-resistant Escherichia coli across China reveals recent endemic plasmid-encoded tet(X4) gene.

Public health interventions to control the recent emergence of plasmid-mediated tigecycline resistance genes rely on a comprehensive understanding of its epidemiology and distribution over a wide range of geographical scales. Here we analysed an Escherichia coli collection isolated from pigs and chickens in China in 2018, and ascertained that the tet(X4) gene was not present at high prevalence across China, but was highly endemic in northwestern China. Genomic analysis of tet(X4)-positive E. coli demonstrated a recent and regional dissemination of tet(X4) among various clonal backgrounds and plasmids in northwestern China, whereas a parallel epidemic coincided with the independent acquisition of tet(X4) in E. coli from the remaining provinces. The high genetic similarity of tet(X4)-positive E. coli and human commensal E. coli suggests the possibility of its spreading into humans. Our study provides a systematic analysis of the current epidemiology of tet(X4) and identifies priorities for optimising timely intervention strategies.

Plasmid-mediated tigecycline-resistant gene tet(X4) in Escherichia coli from food-producing animals, China, 2008-2018.

The recent emergence of plasmid-mediated tigecycline resistance genes, tet(X3) and tet(X4), in animals and humans in China would pose a foreseeable threat to public health. To illustrate this paradigm shift in tigecycline resistance, here, covering the period 2008-2018, we retrospectively analysed a national strain collection of Escherichia coli (n = 2254), obtained from chickens and pigs, in six representative provinces of China. The gene tet(X4) was identified in five pig isolates collected in 2016 and 2018 from the provinces of Sichuan (3/15, 2018), Henan (1/25, 2018) and Guangdong (1/28, 2016), but not in the isolates prior to 2016. None of the isolates was detected harbouring tet(X3). All tet(X4)-positive E. coli exhibited high levels of tigecycline resistance (MICs, 16-64 mg/L), and two were confirmed as colistin resistant, harbouring chromosome-borne mcr-1 gene. The gene tet(X4) was detected on a plasmid in all five isolates, whereas a co-location of tet(X4) on the chromosome of one isolate was observed. Diverse host strains and novel plasmids related to the tet(X4) gene were observed. Our timely findings of the recent emergence of tet(X4) gene in food animal support the rapid surveillance and eradication of this gene before it is established.

Prevalence and Characterization of Fluoroquinolone Resistant Salmonella Isolated From an Integrated Broiler Chicken Supply Chain.

The objectives of this study were to investigate the prevalence and fluoroquinolone resistant Salmonella isolated from an integrated broiler chicken supply chain and their molecular characterization. In total, 73 Salmonella isolates were recovered from a broiler chicken supply chain in Shanghai. Salmonella isolates were tested for susceptibility to 11 antimicrobial agents using the broth dilution method and were characterized using pulsed-field gel electrophoresis (PFGE). Then, the Salmonella isolates were examined for mutations in quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE, and were screened for plasmid-mediated quinolone resistance (PMQR) genes. Lastly, we sequenced the plasmids carrying qnrS1 in six Salmonella isolates from three sources (two isolated per source). Among 73 Salmonella isolates, 45 isolates were identified as S. Indiana, 24 were S. Schwarzengrund, 2 were S. Enteritidis, and 2 were S. Stanleyville. In addition, high rates of resistance were detected for nalidixic acid (41.1%) and ciprofloxacin (37.0%), while resistance to other test agents was diverse (2.0-100%). S. Indiana and S. Schwarzengrund isolates from different sources exhibited the same PFGE pattern, suggesting that the Salmonella isolates possessed high potential to spread along the broiler chicken supply chain. gyrA and parC exhibited frequent missense mutations. Moreover, qnrS1 was the most prevalent PMQR gene in the 73 Salmonella isolates, and it was found about a new hybrid plasmid. This study concludes a high prevalence of fluoroquinolone resistant Salmonella in chicken supply chain, threatening the treatment of Salmonella foodborne diseases. In particular, the emergence of a new hybrid plasmid carrying qnrS1 indicates that the recombination of plasmid carrying resistance gene might be a potential risk factor for the prevention and control strategies of drug resistance.

Distinct mechanisms of acquisition of mcr-1 -bearing plasmid by Salmonella strains recovered from animals and food samples.

Since the report of its discovery in E. coli in late 2015, the plasmid-mediated colistin resistance gene, mcr-1, has been detected in various bacterial species in clinical setting and various environmental niches. However, the transmission mechanisms of this gene in Salmonella is less defined. In this study, we conducted a comprehensive study to characterize the genetic features of mcr-1-positive Salmonella strains isolated from animals and foods. Our data revealed that Salmonella recovered from animals and food specimens exhibited highly different PFGE patterns, and acquired mcr-1-encoding plasmids via different mechanism. Plasmids harboring mcr-1 in Salmonella food isolates were all conjugative and similar as plasmids reported in other species of Enterobacteriaceae, whereas mcr-1-bearing plasmids from animal Salmonella isolates were not conjugative, and belonged to the IncHI2 type. The lack of a region carrying the tra genes was found to account for the inability to undergo conjugation for various sizes of IncHI2 plasmids harbored by animal strains. These data suggest that transmission of mcr-1-positive Salmonella from animal to food might not be a common event and food isolates may have acquired mcr-1-bearing plasmids from other mcr-1-positive bacteria such as E. coli, which co-exist in food samples.

Prevalence and Molecular Characterization of mcr-1-Positive Salmonella Strains Recovered from Clinical Specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ∼30 to ∼250 kb, among which there were conjugative plasmids of ∼30 kb, ∼60 kb, and ∼250 kb and nonconjugative plasmids of ∼140 kb, ∼180 kb, and ∼240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event.

Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

[Study on the functions of potential new genes of Yersinia pestis type three secretion system].

OBJECTIVE: To investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS). METHODS: Mutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A ß-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS. RESULTS: When grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The ß-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain. CONCLUSION: YpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Impact of Hfq on global gene expression and intracellular survival in Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.

Complete genome sequence of Brucella abortus strain BCB034, a strain of biovar 2 isolated from human.

Brucella abortus is divided into eight biovars, of which biovars 1 to 3 are the most frequently represented biovars in strains isolated from humans. Here, we report the genome sequence of B. abortus strain BCB034, a strain isolated from a human patient and that belongs to biovar 2.

Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice.

Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.