Salmonella enterica serotype Virchow: epidemiology, resistance patterns and molecular characterisation of an invasive Salmonella serotype in Israel

This study outlines the unique epidemiology of Salmonella enterica serotype Virchow in Israel. Between 1997 and 2002, the overall incidence of non-typhoid Salmonella enterica (NTS) decreased from 69.3 to 53.3 infections/100,000 population, but the incidence of S. Virchow increased (from 7.2 to 9.1 infections/100,000). Since 2000, S. Virchow has become the second-ranking NTS isolate, accounting for 17% and 27% of all stool and blood NTS isolates, respectively. Infants aged < 1 year had the highest incidence of isolation from stools (92.8/100,000). The incidence of isolation from blood was highest for infants aged <1 year (4.4/100,000). Only 6% of isolates were susceptible to all ten antibiotic agents tested; 34% were resistant to one agent, 54% to one to three agents, and 40% to four to six agents. A high proportion of the tested isolates were resistant to nalidixic acid (89%), streptomycin (56%), tetracycline (43%), trimethoprim-sulphamethoxazole (38%) and chloramphenicol (28%), but none to ciprofloxacin or ceftriaxone. Pulsed-field gel electrophoresis revealed two closely related clusters, each containing a predominant pulsotype. Coupled with its invasive propensity, the increasing incidence of highly resistant S. Virchow in Israel is of real concern. Future research should focus on the sources of S. Virchow in the food chain in order to institute effective control measures.     

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。     

Characterisation of the first CTX-M-10-producing isolate of Salmonella enterica serotype Virchow

Microbiological analysis of a urine sample from an outpatient with symptoms of urinary infection detected >10(5) CFU/mL urine of Salmonella enterica serotype Virchow with resistance to cefotaxime. Molecular analysis demonstrated the presence of the gene encoding CTX-M-10 beta-lactamase in this clinical isolate. This is the first report of this enzyme in Salmonella spp.     

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.     

Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi

Purpose: There are increasing reports on failure of clinical response to ciprofloxacin in typhoid fever despite the strain being sensitive to drug in in-vitro using standard guidelines and showing mutations in DNA gyrase. But this increased MIC and clinical failures with ciprofloxacin are not always co-related with mutations presently identified in gyrA and parC genes. This shows that there may be other mechanisms such as an active drug efflux pump responsible as has been shown in other Enterobacteriaceae. This study was carried out to determine the role of efflux pump in Salmonella Typhi isolates. Materials and Methods: Total 25 already characterized nalidixic acid sensitive and nalidixic acid resistant S. Typhi strains with different range of ciprofloxacin MIC were included to study the role of efflux pump in the presence of CCCP (efflux pump inhibitor). For genotypic characterization, the entire acrR gene was sequenced to confirm the presence of any mutation in the gene. Results: The MIC of ciprofloxacin remained same in the presence and absence of CCCP in the studied strains and no significant mutations were found in the acrR gene in any of the isolates studied. Conclusions: No role of efflux pump in ciprofloxacin resistance was found in strains studied. There is a need to explore further mechanism of ciprofloxacin resistance in Salmonella Typhi.     

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.     

Occurrence of extended-spectrum β-lactamase-producing Salmonella enterica in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans

Among the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum β-lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a bla _SHV-2 gene. The bla _CTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the bla _CTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other bla _CTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals.     

Plasmid-mediated quinolone resistance in typhoidal Salmonellae: A preliminary report from South India

Background: Fluoroquinolones are the drugs extensively employed for the treatment of Salmonella infections. Over the couple of decades that have elapsed since the introduction of fluoroquinolones, resistance to these agents by Enterobacteriaceae family members has become common and widespread. Although fluoroquinolone resistance is mediated by genomic DNA (deoxyribonucleic acid) as well as plasmid DNA, the plasmid-mediated quinolone resistance (PMQR) facilitates higher level resistance by interacting with genomic mechanism and is capable of horizontal spread. Materials and Methods: During a period of 1-year, 63 typhoidal Salmonellae were isolated from 14,050 blood cultures and one parietal wall abscess. 36 (56.25%) were Salmonella Typhi and 27 (42%) were Salmonella Paratyphi A. They were all screened for resistance by the disc diffusion method and their minimum inhibitory concentrations were determined using agar dilution, broth dilution and E-strip method. Ciprofloxacin resistant isolates were screened for PMQR determinants by polymerase chain reaction assay. Results: All the 63 isolates were resistant to nalidixic acid. Among the 36 S. Typhi isolates 20 were resistant to ciprofloxacin, of which 14 carried the plasmid gene qnrB and one carried the aac(6’)-Ib-cr gene. qnrA and qnrS genes were not detected. Ciprofloxacin resistance was not seen in any of the S. Paratyphi A isolates. Conclusion: The antibiotic sensitivity pattern of typhoidal Salmonellae shows an increasing trend of PMQR. The allele B of qnr gene was found to be the predominant cause of PMQR in this study.     

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.     

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.     

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.     

Antimicrobial resistance in Salmonella enterica serovar typhi and paratyphi in South Asia-current status,issues and prospects

Abstract

The human race owes a debt of gratitude to antimicrobial agents, penicillin and its successors that have saved people from tremendous pain and suffering in the last several decades. Unfortunately, this consideration is no more true, as millions of people are prone to the challenging threat of emergence of antimicrobial resistance worldwide and the menace is more distressing in developing countries. Comparable with other bacterial species, Salmonella enterica serovar Typhi (S. typhi) and Paratyphi (S. paratyphi) have been evolving multidrug resistance (MDR) against a wide array of antibiotics, including chloramphenicol, ampicillin and co-trimoxazole, and globally affecting 21 million people with 220?000 deaths each year. S. typhi and S. paratyphi infections are also endemic in South Asia and a series of antibiotics used to treat these infections, have been losing efficacy against enteric fever. Currently, quinolones are regarded as a choice to treat MDR Salmonella in these regions. Travel-related cases of enteric fever, especially from South Asian countries are the harbinger of the magnitude of MDR Salmonella in that region. Conclusively, the MDR will continue to grow and the available antimicrobial agents would become obsolete. Therefore, a radical and aggressive approach in terms of rational use of antibiotics during treating infections is essentially needed.     

Antimicrobial resistance of Salmonella enterica serovar Heidelberg isolated from poultry in Alberta

Salmonella enterica serovar Heidelberg is one of the top three serovars implicated in human infections in Canada. In 2003, the Canadian Integrated Program for Antimicrobial Resistance Surveillance reported antimicrobial resistance (AMR) in S. Heidelberg in Canada. The study objective was to investigate the AMR of S. Heidelberg isolated from poultry in Alberta. We examined 951 S. Heidelberg poultry isolates obtained during 1996 to 2010 and tested against 18 antibiotics using the Sensititre AVIAN1F system. Temporal resistance patterns were analysed using single-level logistic regression models. Continuous variables were included in the multivariable models. Multivariable models were built and variables and interactions were included in these final models. Data were analysed using Stata 11 Intercooled. Ceftiofur resistance ranged annually from 0 to 10.5% and gentamicin resistance ranged annually from 0 to 33.3%; no isolates were enrofloxacin resistant. Resistance to amoxicillin (annual range 0 to 42.6%) varied significantly by time and interaction with commodity type. Meat turkey S. Heidelberg isolates had higher ceftiofur resistance compared with chickens: layers plus layer breeders (odds ratio = 22.6, P < 0.01) and broiler breeders (odds ratio = 9.1, P < 0.01). Gentamicin resistance decreased significantly over the study period (odds ratio = 0.72 per year, P < 0.01). Tetracycline (TET) resistance changed significantly over time (annual range 0 to 39.6%), interacting with poultry commodity type. Meat turkey isolate TET resistance, higher overall than that of chicken, increased throughout the study. All turkey breeder isolates were resistant to TET. In conclusion, this study provides AMR data for S. Heidelberg isolates from the Alberta poultry industry and demonstrated significant trends in resistance, both temporal and between poultry commodities.     

萘啶酸抗性甲型副伤寒病原菌的克隆扩散和遗传多样性

目的 认识肠沙门菌甲型副伤寒血清型(SPA)的克隆扩散和遗传多样性,建立并确定病原菌流行克隆的分型方法.方法 采用有对照的K-B纸片扩散技术对分离的3980株SPA进行抗微生物药物敏感性试验;经PCR扩增和基因测序检测15个萘啶酸抗性菌株喹诺酮抗性决定区的gyrA、gyrB、syrC和syrE基因;采用Spe Ⅰ和Xba Ⅰ消化染色体DNA脉冲场凝胶电泳(PFGE)对来自7个县的121个分离株进行分型和聚类分析.结果 萘啶酸敏感菌株在1999年占有优势,但2000年以后被萘啶酸抗性菌株替代;15个萘啶酸抗性菌株的PCR扩增和基因测序显示抗性机制是由gyrA基因的单点突变引起;121个菌株spe Ⅰ和Xba Ⅰ消化产物分别得出以Spe Ⅰ 01、spe Ⅰ 02或Xba Ⅰ 01型占优势的5种和4种PFGE型,Spe Ⅰ 01和Spe Ⅰ 02分别占37.2%和57.9%,或Xhn Ⅰ 01占95.0%.结论 在研究期间SPA分离株萘啶酸抗性率上升;PFGE型的SpeⅠ01和SpeⅠ 02或XbaⅠ01是玉溪流行的主要克隆;采用Spe Ⅰ和Xba Ⅰ的PFGE是鉴别SPA的一项有用技术.     

Treatment of enteric fever in children on the basis of current trends of antimicrobial susceptibility of Salmonella enterica serovar typhi and paratyphi A

Purpose: Recent reports indicate decreased susceptibility of S. typhi to fluoroquinolones, especially ciprofloxacin. Chloramphenicol has been suggested as first line therapy of enteric fever in many studies. This is a prospective study that describes the trends of antimicrobial susceptibility of S. typhi and S. paratyphi A causing bacteraemia in children and reports therapeutic failure to ciprofloxacin and evaluates the possible use of chloramphenicol, ampicillin, ciprofloxacin and third generation cephalosporins as first line therapy in the treatment of enteric fever in children. Methods: The present study was conducted from April 2004 to March 2005 in a superspeciality children hospital at New Delhi. A total of 56 S. typhi and five S. paratyphi A isolates were obtained among the 673 blood cultures performed. Antimicrobial testing was done using disk diffusion technique (NCCLS method) for 13 antimicrobials and MICs were calculated for ampicillin, ciprofloxacin, chloramphenicol and cefotaxime. Analysis of data was done using WHONET software. Results: All 56 isolates of S. typhi were sensitive to amoxycillin+clavulanate, gentamicin, cefixime, cefotaxime and ceftazidime. Multidrug resistance (MDR, resistance to three drugs) was seen in 22 cases (39%) and resistance to five drugs was seen in 12 cases (21%). Only two isolates were resistant to chloramphenicol (3%). MIC₉₀ for ampicillin, chloramphenicol, ciprofloxacin and cefotaxime were 1.0 μg/ml, 4.0 μg/ml, 64 μg/ml and 0.125 μg/ml respectively. All S. paratyphi A isolates were sensitive to ampicillin and chloramphenicol and resistant to nalidixic acid.MIC distribution data for chloramphenicol revealed elevated MIC but still in susceptible range. Conclusions: There is an urgent need for further clinical studies to evaluate response to chloramphenicol in such cases. Antimicrobial susceptibility data and MIC distribution favour use of ampicillin as a drug of choice for the treatment of enteric fever. Third generation cephalosporins are also useful but their use should be restricted for complicated cases.     

Molecular characterisation of multidrug-resistant Salmonella enterica serovar Typhimurium isolates from Gomel region, Belarus

This study describes the characterisation by pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA) typing and antimicrobial resistance profiles of 35 Salmonella enterica serovar Typhimurium isolates, mostly from infections in children who acquired an infection outside hospitals in the Gomel region of Belarus. Thirty-one isolates were highly similar according to PFGE and MLVA typing, were multidrug-resistant, including resistance to ceftiofur, and harboured the bla(CTX-M-5) gene. These results indicate that a common source may have been responsible for most of the infections.     

High prevalence of ceftriaxone resistance among invasive Salmonella enterica serotype Choleraesuis isolates in Thailand: The emergence and increase of CTX-M-55 in ciprofloxacin-resistant S. Choleraesuis isolates

S. Choleraesuis is a highly invasive zoonotic pathogen that causes a serious systemic infection in humans. The emergence and increase of resistance to ceftriaxone and ciprofloxacin among S. Choleraesuis has become a serious therapeutic problem. The present study demonstrated high frequency of antimicrobial resistance in Salmonella Choleraesuis among 414 nontyphoidal Salmonella isolates from bacteremic patients in Thailand. High rates of ceftriaxone (58.3%) and ciprofloxacin (19.6%) resistances were observed in S. Choleraesuis isolates. The dissemination of the self-transferable bla_CTX-M-14-carrying IncFII_s, IncFII, and IncI1 plasmids and bla_CMY-2-carrying IncA/C plasmid along with the clonal spread of bla_CMY-2-harbouring S. Choleraesuis isolates contributed to the high frequency of resistance to extended-spectrum cephalosporins (ESCs; third- and fourth-generation cephalosporins) during 2005–2007. We reported the first occurrence of ceftazidime-hydrolysing CTX-M-55 in S. Choleraesuis isolates which dramatically increased and became the most abundant CTX-M variant among ESC-resistant S. Choleraesuis isolates during 2012–2016. The spread of clone pulsotype B3 was due to the dissemination of IncA/C plasmids carrying both bla_CTX-M-55 and qnrS1 among ciprofloxacin-resistant S. Choleraesuis isolates harbouring D87G in GyrA. These isolates were apparently responsible for the high rates of co-resistance to ESCs and ciprofloxacin (51.3%) during 2012–2016. This study emphasizes the importance to have an action plan to control the dissemination of antimicrobial resistance in S. Choleraesuis since this poses a threat to global health due to travel and trade in animal food products.     

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.