Antimicrobial resistance and R-factor transfer of salmonellae isolated from chicken carcasses in Greek hospitals

A total of 62 salmonellae, belonging to six different serotypes, were isolated from 60 out of 87 (69.0%) chicken carcasses delivered to hospitals of Thesssaloniki, Greece. Salmonella enteritidis, Salmonella anatum and Salmonella bredeney were the most prevalent serovars. Isolates were examined for antibiotic resistance patterns and R-determinants. Resistance to at least one antibiotic was observed in 36 (58.1%) of them and 18 different resistant profiles were recorded. Nitrofurantoin-resistance was the most common (29.0%), followed by spectinomycin (21.0%), ampicillin (19.4%) and ticarcillin (19.4%). Fourteen (38.9%) of the resistant isolates possessed R-factors and resistance to ampicillin, ticarcillin, trimethoprim and kanamycin was easily self-transferable. However, nitrofurantoin- and spectinomycin-resistance although prevailing, was not found transferable even after mobilization. The high incidence of antibiotic resistant salmonellae among chicken carcasses in our hospital setting suggests the need for public health interventions and possible withdrawal of drug selective pressure.     

肠道杆菌的扫描电镜观察

我们用扫描电镜观察肠道杆菌种(Enterobacteriaceae)中的四种细菌,大肠埃希氏杆菌(Escherichiacoli),普通变形杆菌(Proteus vulgaris),伤寒沙门氏菌(Salmonella typhi)和福氏志贺氏菌(Shigella Flexneri)。未处理的菌落标本,菌落表面都可形成一层厚薄不同的表膜(Surface film)。四种细菌菌落的表膜,形状不一,千姿百态。适当处理后的菌落标本,则可显示菌体的本来面目。四种细菌菌体在菌落表面的分布和排列,也是各不相同,千差万别。本文根据扫描电镜的观察,对四种不同菌落进行了讨论。     

Recent Advances in the Detection of Antibiotic and Multi-Drug Resistant Salmonella: An Update

Antibiotic and multi-drug resistant (MDR) Salmonella poses a significant threat to public health due to its ability to colonize animals (cold and warm-blooded) and contaminate freshwater supplies. Monitoring antibiotic resistant Salmonella is traditionally costly, involving the application of phenotypic and genotypic tests over several days. However, with the introduction of cheaper semi-automated devices in the last decade, strain detection and identification times have significantly fallen. This, in turn, has led to efficiently regulated food production systems and further reductions in food safety hazards. This review highlights current and emerging technologies used in the detection of antibiotic resistant and MDR Salmonella.     

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes,the Basis of Its Specificity and Bactericidal Activity

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide–membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium^2GN, Escherichia coli^3GN, and Staphylococcus aureus^3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.     

Correlation between CRISPR Loci Diversity in Three Enterobacterial Taxa

CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains).     

Characterization of Two-Component System CitB Family in Salmonella Pullorum

Salmonella enterica, serovar Gallinarum, biovar Pullorum, is an avian-specific pathogen which has caused considerable economic losses to the poultry industry worldwide. Two-component systems (TCSs) play an essential role in obtaining nutrients, detecting the presence of neighboring bacteria and regulating the expression of virulence factors. The genome analysis of S. Pullorum strain S06004 suggesting the carriage of 22 pairs of TCSs, which belong to five families named CitB, OmpR, NarL, Chemotaxis and LuxR. In the CitB family, three pairs of TCSs, namely CitA-CitB, DcuS-DcuR and DpiB-DpiA, remain unaddressed in S. Pullorum. To systematically investigate the function of the CitB family in S. Pullorum, four mutants, ΔcitAB (abbreviated as Δcit), ΔdcuSR (Δdcu), ΔdpiBA (Δdpi) and ΔcitABΔdcuSRΔdpiBA (Δ3), were made using the CRISPR/Cas9 system. The results demonstrated that the CitB family did not affect the growth of bacteria, the results of biochemical tests, invasion and proliferation in chicken macrophage HD-11 cells and the expression of fimbrial protein. But the mutants showed thicker biofilm formation, higher resistance to antimicrobial agents, enhanced tolerance to inhibition by egg albumen and increased virulence in chicken embryos. Moreover, the deletion of Dpi TCS was detrimental to survival after exposure to hyperosmotic and oxidative environments, as well as the long-term colonization of the small intestine of chickens. Collectively, we provided new knowledge regarding the possible role of the CitB family involved in the pathogenic processes of S. Pullorum.     

大黄抑制食源性致病菌的活性成分研究

研究了大黄对大肠杆菌、沙门氏菌和金黄色葡萄球菌的抑菌效果,初筛的结果显示大黄醇提物对这3种菌均有较强的抑菌作用,并发现其中抑菌作用最强的物质分布在大黄氯仿段萃取物中。利用UPLC-MS-MS分析大黄氯仿段萃取物,发现大黄氯仿段萃取物的主要成分分别是大黄素、芦荟大黄素、大黄酸、大黄素甲醚和大黄酚。在这五种化合物中,大黄酸对大肠杆菌和沙门氏菌具有最强的抑菌效果,对大肠杆菌和沙门氏菌的最小抑菌浓度分别为125μg/m L和250μg/m L,最小杀菌浓度分别为250μg/m L和500μg/m L,大黄素对于金黄色葡萄球菌具有较强的抑菌效果,最小抑菌浓度和最小杀菌浓度分别为62.5μg/m L和125μg/m L。      

雾化植物精油对鼠伤寒沙门氏菌抑制效果和樱桃番茄品质的影响

本研究的目的是评测雾化液态植物(芥末、肉桂、牛至和百里香)精油对樱桃番茄表面和茎端的抑菌效果及对果实品质的影响。将樱桃番茄的表面和茎端分别接种鼠伤寒沙门氏菌(ATCC 53467和ATCC 5 3468菌种),晾干之后放到包装盒中,采用加湿器来雾化液态的植物精油,然后通过一根PE管导入到装有已经接种了鼠伤寒沙门氏菌的樱桃番茄的包装盒中。处理后的果实存放在10℃的冰箱中3个星期,存放期间每隔1个星期进行微生物指标测定,不接菌种的果实用来测定果实品质指标。结果表明,雾化的植物精油能够显著的抑制樱桃番茄表面和茎端的鼠伤寒沙门氏菌(表面及茎端菌落分别降低2.00 log CFU/fruit和1.00 log CFU/fruit左右)。随着贮藏期的延长,菌落总数也随之降低,这证明其抑菌效果仍然有效。雾化植物精油杀菌处理对果实硬度,颜色,VC等品质指标没有显著影响(p>0.05)。雾化是一种方便有效的可用于储存或运输果蔬产品消毒的新方法。      

免疫磁捕获-荧光定量PCR快速检测食品中沙门氏菌

建立了免疫磁珠分离（Immunomagnetic separation,IMS）联合荧光定量PCR（Real-time PCR）技术准确、快速检测食品中沙门氏菌（Salmonella spp）的方法。采用亲和纯化的羊抗沙门氏菌抗体与Dynabeads M-280磁珠制备沙门氏菌免疫磁珠,优化反应条件,建立免疫磁珠分离方法。同时根据沙门氏菌特异性ttr基因,建立Real-time PCR体系,并检测其特异性及敏感性。结果显示,沙门氏菌可产生荧光信号,而其他细菌均未见明显荧光信号。利用所建立的免疫磁捕获-荧光定量PCR（IMS-real-time PCR）方法可以检测初始含菌量最低为6.5CFU/25g的食品样品,全过程需时约8h。150份实际食品样品中,IMS-real-time PCR方法检出27份阳性样品,免疫磁珠联合XLT4平板培养法检出18份阳性样品,传统国标法检出14份阳性样品。      

Expression profiles of effector proteins SopB, SopD1, SopE1, and AvrA differ with systemic, enteric, and epidemic strains of Salmonella enterica

The presence and expression of sopB, sopD1, sopE1, and avrA genes encoding virulence associated effector proteins were studied comparatively in 405 Salmonella enterica strains. They belong to different serovars and clonal types (genotypes, phage types) and originated from different clinical (systemic infection, focal enteritis, enterocolitis) and epidemic sources (epidemics, sporadic cases). The sopB and sopD1 determinants were commonly prevalent, but sopE1 and avrA genes only in 55% and 80%, respectively. A correlation of this pattern of absence and presence of the respective genes to the epidemic and clinical origin could not be detected. In contrast, the expression of the respective genes appeared differently: SopB and SopE1 proteins are well produced, but SopD1 and AvrA proteins only rarely under the applied standard culture conditions. However, using a range of different environmental signals (temperature, pH, cations, etc.) some of the S. enterica nonproducer strains (e. g., S. Agona, S. Bovismorbificans, S. Virchow, etc.) begin to produce AvrA and SopD1. They turned now into an expression profile which was found typically for the epidemic strains of S. Typhimurium and S. Enteritidis. Also S. enterica strains from systemic infections could be characterized by their strong SopB and SopE1 expression while SopD1 and AvrA proteins were missing. Although it is premature to outline generally a correlation of these expression profiles and the clinical and epidemiological potency of Salmonellae, the reported results allow a first understanding how a fine tuning of their virulence will take place.