Molecular epidemiology of ampicillin-resistant clinical isolates of Salmonella enterica serovar Typhimurium

Thirty-nine multiresistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates were obtained from 33 children and 6 adults hospitalized from 1996 to 1999 in the University Hospital of Amiens (France). S. Typhimurium was cultured from stools (n=36), blood samples (n=2) and peritoneal fluid (n=1). These isolates were characterized by biotyping, antibiotic susceptibility test, RAPD-PCR, and PFGE typing. Emergence of pentaresistant S. Typhimurium isolates (phenotype ACSSuTe) was observed, and five of them were resistant to nalidixic acid and of intermediate susceptibility to pefloxacin. Genotypic analysis of both RAPD and PFGE results showed that there were 7 different patterns. Thirty-three isolates gave an identical pattern (AI) and were considered as epidemic isolates; the six remaining patterns (each containing one isolate) corresponded to sporadic cases. Antibiotic susceptibility patterns, RAPD and PFGE patterns subdivided the 39 isolates into 9 clonally related groups. One of them (pattern AI and R-pattern a) was implicated in 74% of the cases.     

一株不典型山夫登堡沙门氏菌的鉴定及其16SrRNA序列分析

目的对一株从广州市食品从业人员肛拭子中分离的蔗糖发酵型沙门氏菌进行鉴定。方法应用培养特性试验、生化分析、血清型鉴定,及16S rRNA序列分析进行鉴定。结果该菌培养特性及生化特性符合沙门氏菌定义,但与普通沙门氏菌在蔗糖利用、硫化氢产生上存在差异。血清型鉴定为山夫登堡沙门氏菌（Salmonella enterica Subsp·enterica serovar Senftenberg）,16S rRNA序列分析为肠道沙门氏菌亚种（Salmonella enterica subsp）。结论该菌为一株蔗糖发酵型、硫化氢阴性的不典型山夫登堡沙门氏菌。     

Salmonella enterica serovar Typhi plasmid pRST98-mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts

pR_ST98 is a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi) and mediates both drug-resistance and virulence of S. typhi. Autophagy has been recently reported as an important component of the innate immune response against intracellular pathogen. In this study, we investigated the effect of pR_ST98 on cellular autophagy, apoptosis and bacterial survival in infected fibroblasts. S. typhi strain ST₈ carrying pR_ST98, Salmonella typhimurium strain SR-11 carrying a 100 Kb virulent plasmid, and avirulent S. typhi strain ST₁₀ without plasmid were tested in this experiment. Results showed that embryonic fibroblasts infected with ST₈ containing pR_ST98 had decreased autophagy accompanied by increased bacterial survival and apoptosis. Further study showed that autophagy inducer rapamycin reversed pR_ST98-mediated inhibition of autophagy and reduced apoptosis in infected fibroblasts. Our data indicate that pR_ST98 can inhibit autophagy, thus facilitating S. typhi survival and promoting apoptosis of host cells. This study contributes to understanding the underlying mechanism of pR_ST98-mediated virulence in S. typhi.     

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90?fg/μl or 10² CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.     

Contribution of the csgA and bcsA genes to Salmonella enterica serovar Pullorum biofilm formation and virulence

Salmonella biofilm formation is important to environmental stress resistance and virulence. However, the roles of the csgA and bcsA genes, which affect curli protein and cellulose production, respectively, in Salmonella enterica serovar Pullorum, are unknown. Here we constructed deletions in the csgA and bcsA genes in S. enterica serovar Pullorum strain S6702 and evaluated several aspects of biofilm formation and virulence. ΔcsgA showed decreased production of curli fimbriae, while ΔbcsA had reduced cellulose production. Both mutants had a reduced ability to form biofilms. ΔcsgA was reduced in adhesion and invasion to HeLa cells and exhibited decreased intracellular proliferation in HD11 macrophages. ΔbcsA exhibited increased proliferation in HD11 cells and replicated better in chicken spleens, as compared to the wild-type strain. ΔcsgA virulence was attenuated in assays involving oral challenge of one-day-old chickens.     

Global regulators and environmental adaptation in Gram-negative pathogens

A powerful combination of single-gene studies and whole genome approaches has provided a wealth of information about the regulatory circuits used by bacteria to adapt to the environmental changes that are encountered during infection. The facultative intracellular pathogen Salmonella enterica will be used to illustrate how global regulators such as the nucleoid-associated proteins Fis and H-NS collaborate with fluctuations in the superhelicity of the DNA template to modify the gene expression profile of the bacterial cell outside and inside the host.     

Construction and characterization of a cigR deletion mutant of Salmonella enterica serovar Pullorum

Salmonella enterica serovar Pullorum (S. Pullorum) is the causative agent of pullorum disease (PD) and results in severe economic losses to the poultry industry. As a Salmonella type III secretion system 2 (T3SS2) effector and predicted membrane protein, CigR is encoded by the cigR gene within Salmonella pathogenicity island 3 (SPI3). In order to research the influence of the cigR gene on S. Pullorum, a cigR mutant of S. Pullorum S06004 was constructed by the lambda Red recombination system, and then its characterization was analysed. Lack of cigR did not affect the growth and biochemical properties, but resulted in decreased biofilm formation. The mutant strain was stable with the deletion of the cigR gene. Macrophage infection assay and in vivo competition assay showed that the mutant strain increased the replication and/or survival ability in the HD11 cell line and in chickens compared to that of the parent strain, the median lethal dose (LD₅₀) of the mutant strain was one-fifth of the parent strain for 2-day-old chickens when injected intramuscularly. These results demonstrate CigR plays roles in biofilm formation and pathogenicity of S. Pullorum, deletion of cigR can significantly decrease biofilm formation and significantly increase virulence.     

Yin and Yang of Biofilm Formation and Cyclic di-GMP Signaling of the Gastrointestinal Pathogen Salmonella enterica Serovar Typhimurium

Within the last 60 years, microbiological research has challenged many dogmas such as bacteria being unicellular microorganisms directed by nutrient sources; these investigations produced new dogmas such as cyclic diguanylate monophosphate (cyclic di-GMP) second messenger signaling as a ubiquitous regulator of the fundamental sessility/motility lifestyle switch on the single-cell level. Successive investigations have not yet challenged this view; however, the complexity of cyclic di-GMP as an intracellular bacterial signal, and, less explored, as an extracellular signaling molecule in combination with the conformational flexibility of the molecule, provides endless opportunities for cross-kingdom interactions. Cyclic di-GMP-directed microbial biofilms commonly stimulate the immune system on a lower level, whereas host-sensed cyclic di-GMP broadly stimulates the innate and adaptive immune responses. Furthermore, while the intracellular second messenger cyclic di-GMP signaling promotes bacterial biofilm formation and chronic infections, oppositely, Salmonella Typhimurium cellulose biofilm inside immune cells is not endorsed. These observations only touch on the complexity of the interaction of biofilm microbial cells with its host. In this review, we describe the Yin and Yang interactive concepts of biofilm formation and cyclic di-GMP signaling using S. Typhimurium as an example.     

Construction of pSPI12-cured Salmonella enterica serovar Pullorum and identification of IpaJ as an immune response modulator

In Salmonella, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the ipaJ gene in a multi-copy plasmid pSPI12 prevalent in Salmonella enterica serovar Pullorum (S. Pullorum), we established a method to eliminate the plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 by using the suicide vector pDM4. Briefly, a 500?bp fragment ipaJU from pSPI12 was cloned into pDM4 and transformed into S. Pullorum C79-13 by conjugative transfer. After homologous recombination, the suicide vector was inserted into pSPI12 to produce pSPI12-pDM4-ipaJU. Induction of the expression of the sacB gene in the suicide vector killed the bacteria harbouring plasmid, while the progeny losing the plasmid survived in the plate with sucrose. The plasmid-cured strain showed extremely decreased ability to infect chicken macrophage HD11 cells and LMH hepatic epithelial cells compared to wild type strain and complementary strain carrying ipaJ. Additionally, IFN-γ mRNA levels were up-regulated in HD11 cells or chicken spleens infected by plasmid-cured strain, but no difference was detected in IL-4 among the three strains. Transforming ipaJ into S. Enteritidis also decreased expression of proinflammatory cytokines in infected macrophages or chicken spleens compared to wild type strain. These results suggest that the ipaJ gene in pSPI12 is involved in S. Pullorum infection and that IpaJ protein modulates immune response.     

Intracellular growth and survival of Salmonella enterica serovar Typhimurium carrying truncated hemoglobins of Mycobacterium tuberculosis

Two distantly related truncated hemoglobins (trHbs), HbN and HbO, are produced at different growth stages of Mycobacterium tuberculosis. Oxygen and nitric oxide (NO) binding properties of these trHbs suggest their vital role(s) in adaptation of tubercle bacillus under hypoxic and nitrosative stress conditions. Here, we have demonstrated that HbN of M. tuberculosis provides distinct advantage over HbO in supporting intracellular growth and survival of the heterologous host, Salmonella enterica serovar Typhimurium, during macrophage infection specifically against toxicity of NO. HbN and HbO encoding genes of M. tuberculosis have been expressed in a NO-sensitive hmp mutant of S. enterica serovar Typhimurium that exhibits attenuated growth within the macrophages. Presence of HbN and HbO conferred distinct oxygen dependent NO metabolizing activity to the mutant S. enterica serovar Typhimurium. However, the HbN carrying cells exhibited nearly 2-3-fold higher NO metabolizing activity than the isogenic strain having HbO under aerobic condition. More than half of the NO uptake activity of HbN carrying cells was retained when oxygen level dropped to microaerobic condition. In comparison, NO uptake activity of HbO carrying cells of mutant S. enterica dropped drastically (90%) under similar hypoxic conditions. When internalized by mice peritoneal macrophages, HbN carrying cells exhibited 3- and 4-fold higher survival compared to similarly bound and internalized HbO carrying and control cells, respectively. The protective effect of HbN persisted even after activation of macrophages in the presence of IFN-gamma, whereas, HbO did not show any significant effect on survival of the NO-sensitive hmp mutant of Salmonella. These results provide strong experimental evidence in support of the protective role of HbN against nitrosative stress inside macrophages and suggest that intracellular protection conferred by HbN of M. tuberculosis might not be restricted to its native host only.     

Differences in the carriage and the ability to utilize the serotype associated virulence plasmid in strains of Salmonella enterica serotype Typhimurium investigated by use of a self-transferable virulence plasmid, pOG669

Most strains of Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) naturally harbour a virulence plasmid which carries the salmonella plasmid virulence (spv) genes. However, isolates belonging to certain phage types are generally found without the plasmid. We have utilized a self-transferable virulence plasmid, pOG669 to investigate the effect of introduction of spv genes into strains of such phage types. The use of the co-integrate plasmid, pOG669, was validated on a diverse collection of strains. pOG669 was transferred into strains of serotypes that are normally associated with the possession of virulence plasmids. All strains maintained the wild type level of virulence in a mouse model, except that introduction of pOG669 restored normal virulence levels in an avirulent, plasmid free strain of S. dublin and resulted in a decrease in virulence in a strain of S. dublin from clonal line Du3. S. gallinarum did not become virulent in mice, but pOG669 was functionally interchangeable with the wild type plasmid when strains were tested in a chicken model. Strains of serotypes not normally associated with the carriage of a virulence plasmid did not increase in virulence upon the introduction of pOG669. An IncX plasmid pOG670 that was included as control was incompatible with the virulence plasmid in a strain of S. dublin, demonstrating that the common virulence plasmid of this serotype is of a different incompatibility group than other virulence plasmids. Strains of S. typhimurium from phage types that do not normally carry a virulence plasmid responded differently to attempts to introduce pOG669. No transconjugants were observed with the strains of DT5 and DT21. The introduction of pOG669 did not alter the virulence of JEO3942(DT10), DT35 and JEO3949(DT66) significantly, while DT1 and DT27 became more virulent. DT27 became as virulent as wild type C5, while logVC(10) of DT1 only increased from 4.1 to 5.7. The ability to express spv-genes was measured by use of an spvRAB'-cat fusion. Expression in S. enteritidis was found to be higher than in other serotypes tested. Only serotypes that naturally carry a virulence plasmid expressed spv-genes. The strain of DT1 expressed spv at a very low level, while expression in the strains of DT10 and DT35 was approximately 2-fold lower than in a control strain of S. typhimurium, while the level in the DT66 strain corresponded to the control strain. The plasmid pSTF9, which carried the fusion gene could not be introduced into the strains of DT5, DT21 and DT27. The RpoS level in the strains was measured indirectly by use of a katE-lacZ fusion. In the DT5 strain the level of expression was low, while the strains JEO3942(DT10), DT21, DT27 and DT35 expressed 4-5 fold the level in this strain. An internal fragment of the rpoS gene was sequenced in three strains. These all showed an identical sequence to a published S. typhimurium rpoS gene.