Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp.

The distribution and phenotypic activity of the genes encoding for serine protease, glycerophospholipid-cholesterol acyltransferase, lipases, aerolysin/hemolysin and DNases were investigated in 234 isolates identified by 16S rDNA-RFLP representing all the species of Aeromonas. The former three genes were found to be highly conserved among the genus. Aerolysin/hemolysin and DNase genes and β-hemolytic activity were significantly more frequent in clinical than in environmental isolates. Aerolysin/hemolysin and serine protease genes were present in all β-hemolytic strains supporting serine protease as possibly important for the activation of the former gene. The high prevalence of virulence factors in clinical isolates indicates that they may play a role in the mechanisms of pathogenesis of these microorganisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Measurement of virulence of aeromonads using a suckling mouse model of infection

Abstract Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD₅₀) 43 h after oral administration of live bacteria. The LD₅₀ of virulent Aeromonas isolates ranged from log₁₀ 7.53 (mean) organisms to log₁₀ 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD₅₀ and the source of the isolate, β-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.     

High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil

Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer ⁺/aerA⁺/hly ⁺/ast ⁺/alt ⁺ was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly ⁺ (85%) and aerA⁺ (78.7%), while in A. caviae complex strains was aerA⁺ (32.8%). All A. veronii complex sobria strains were aer ⁺/aerA⁺. All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.     

Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes

Aeromonas hydrophila strains recovered from clinical samples and ambient sources were phenotypically and genetically identified. In addition, the distribution of putative virulence factors was assayed. To determine the genetic diversity of these strains, random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers were used. The discriminatory ability of the techniques, using Simpson's index, was 0.96 for both methods. The most consistent dendrogram was obtained when RAPD and ERIC data were combined. The genetic diversity revealed a high intra-specific genetic diversity (h=0.364+/-0.024 and I=0.538+/-0.030). The strains showed a tendency to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). The present study demonstrates and quantifies the high intra-specific diversity within this species and reveals a clear differentiation of strains according to their ecological origin. The distribution of virulence-related genes confirm that A. hydrophila is a genetically heterogeneous species that harbour ecotypes which have different pathogenic potential to human and other animals.     

嗜水气单胞菌若干毒力因子同时缺失的突变株的构建及特性分析

以携带质粒pAM12 0 (Tcr Tn916 )的大肠杆菌CG12 0株为供体菌 ,采用滤膜接合法与受体菌嗜水气单胞菌J_1株 (cfzr)进行接合转移 ,在含Tc和cfz选择平板上进行筛选。共获接合转移菌落 380 0个 ,其接合频率为 3× 10 - 5(按供体细胞计算 )。任取 38个接合子 ,提取基因组DNA ,以嗜水气单胞菌特异性 16SrDNA引物进行PCR扩增 ,所有接合子均阳性。为证明Tn916确实插入基因组 ,以四环素基因 (tet)引物进行PCR扩增 ,结果所有抗性接合子均扩增出一条特异条带。与亲本J_1株相比 ,所有接合子的主要毒力因子如蛋白酶、溶血素、DNA酶和淀粉酶等均不表达 ,对小鼠失去致病力 ,其LD50 大于 10 9CFU。接合子连传 10次后 ,四环素抗性消失 ,但毒力未恢复 ,说明通过转座子Tn916的插入可获得稳定的无毒嗜水气单胞菌突变株。Tn916引起嗜水气单胞菌毒力性状改变的机制有待研究 ,推测可能与该菌染色体上存在Tn916的热点或毒力岛有关。     

Detection of metallo-β-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims:  To determine the prevalence and expression of metallo-β-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil.
Methods and Results:  Eighty - seven Aeromonas isolates belonging to Aeromonas hydrophila ( n  =   41) and Aer. jandaei ( n  =   46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97·6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla _IMP , bla _VIM and bla _SPM-1 were not detected. On the other hand, production of MBL activity was detectable in 87·8% and 10·9% of the cphA -positive Aer. hydrophila and Aer. jandaei isolates respectively.
Conclusions:  Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity.
Significance and Impact of the Study:  These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.     

Biotyping and virulence factors in clinical and environmental isolates of Aeromonas species.

Biochemical characteristics and virulence factors were compared in 147 Aeromonas spp. isolated from patients with diarrhea and in 94 strains isolated from metropolitan water supplies in the same area during the same period. Fermentation of arabinose occurred with 58.5% of the environmental strains and 15% of the clinical isolates; 39.4% of the strains from water and 6.8% of the fecal isolates fermented salicin. The frequency of esculin hydrolysis was the same in both groups. Ninety-one percent of clinical isolates and 70.2% of environmental strains were enterotoxigenic and, except for four clinical isolates, all of these strains also produced hemolysins. Hemagglutination that was inhibited by fucose and mannose but not by galactose was found in 67% of the water isolates and 10.2% of the clinical strains. Although the distribution of several characteristics differs in clinical and environmental strains, many of the strains found in water have properties identical with those of the clinical isolates. We suggest that such strains may be potential enteric pathogens.     

2016年辽宁省婴幼儿配方食品及谷类辅助食品中蜡样芽胞杆菌毒力基因的鉴定

目的 了解婴幼儿配方食品和谷类辅助食品中蜡样芽胞杆菌的毒力基因携带特点,对辽宁省婴幼儿配方食品和谷类辅助食品中蜡样芽胞杆菌的污染状况进行调查。方法 依据GB4789.14‒2014《食品安全国家标准 食品微生物学检验 蜡样芽胞杆菌检验》及采用PCR扩增技术和血平板检测的方法对2016年采自辽宁省15个监测点,收集的176份乳源性食品中检出的22株蜡样芽胞杆菌进行10种毒力基因检测。结果 婴幼儿配方食品和谷类辅助食品蜡样芽胞杆菌检出率为12.5%（22/176）,非溶血性的肠毒素Nhe基因、溶血素BL基因、肠毒素T基因和细胞毒素K基因是辽宁省乳源性蜡样芽胞杆菌的主要毒力基因,至少携带2种毒力基因的菌株达到检出菌总数的100.0%。结论 研究结果证实辽宁省婴幼儿配方食品及谷类辅助食品存在蜡样芽胞杆菌污染情况,严格监控婴幼儿配方食品及谷类辅助食品的蜡样芽胞杆菌污染,对于生产出优质婴幼儿配方食品及谷类辅助食品具有重要意义,以期提高婴幼儿食品的质量安全。     

Distribution of virulence genes in Aeromonas spp. isolated from Sardinian waters and from patients with diarrhoea

AIMS: To characterize 46 isolates of different Aeromonas spp. strains (26 Aeromonas hydrophila, 13 Aeromonas sobria and 7 Aeromonas salmonicida) isolated from coastal water and clinical sources in Sardinia, Italy. METHODS AND RESULTS: The isolates were analysed for the production of the following virulence properties: slime, haemolysin, gelatinase and protease production, and adhesion to eucaryotic epithelial cells. The presence of known virulence genes: A. hydrophila cytolytic enterotoxin gene AHCYTOEN; type IV pilus gene Tap; Bundle forming pilus genes BfpA and BfpG were investigated using polymerase chain reaction (PCR). In addition the enterobacterial repetitive intergenic consensus sequences (ERIC)-PCR fingerprinting was used to further differentiate the strains. CONCLUSIONS: This study confirms the presence of virulent Aeromonas strains in the Mediterranean sea. The study also found a greater prevalence of haemolysin, protease and gelatinase production, as well as a higher adhesion capacity, among strains isolated from patients with diarrhoea. SIGNIFICANCE AND IMAPCT OF THE STUDY: This is the first time that Aeromonads have been isolated and characterized from Sardinian waters and from patients with diarrhoea in Sardinia. This study adds to our knowledge of the ecology of this micro-organism and may in the future help prevent infections both in fish and in humans.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Comparison of lethalities in mouse versus goldfish for clinical and food isolates ofAeromonas hydrophila

Summary The lethality of 16 clinical or food isolates ofAeromonas hydrophila was assessed by determination of LD₅₀ (i.p.) in mice and goldfish. In mice LD₅₀ values for the variousA. hydrophila strains were similar, ranging from 1.2–21.0×10⁸ cells/animal. A wider range of LD₅₀ values, 0.03–11.8×10⁸ cells/animal, was observed with goldfish. Lethality was not correlated between the two test animals. Further, cytotoxic response in Y-1 adrenal cells did not correlate with lethality in either test animal. It appears that lethality is not a good measure of potential enterotoxigenicity, but may be useful in assessing the invasive character of isolates causing systemic infections in immunocompromised hosts.     

临床分离志贺菌中CRISPR/Cas系统的分布及其与毒力基因的关系

【目的】了解临床分离志贺菌中CRISPR/Cas系统的分布特征并分析其与毒力基因的关系。【方法】以聚合酶链式反应(PCR)方法,采用10对引物分别对57株临床分离志贺菌中CRISPR1、cas2-cas1、cas6e-cas5、cas7、cse2、cse1-cas3基因和毒力基因ipaH、ial、ipaBCD、virA进行检测。对CRISPR1的PCR结果进行测序,并用CRISPR finder在线软件对CRISPR1基因座进行分析。通过卡方检验初步分析CRISPR/Cas系统与毒力基因的关系。【结果】测序结果显示,CRISPR1基因座中间隔序列数目较少且在不同菌株间一致性较高;57株志贺菌中,84.2% (48/57)的志贺菌中可检测到CRISPR/Cas系统,其中68.8% (33/48)的志贺菌中cas6e-cas5基因或(和) cse2基因中发现插入序列;毒力基因ipaH、ial、virA、ipaBCD的检出率依次为100%、100%、98.2%和87.7%;毒力基因ipaBCD的阳性率与活性CRISPR/Cas系统的分布无关(P>0.05)。【结论】CRISPR/Cas系统广泛存在于临床分离志贺菌中;部分cas基因中有插入序列;并未发现志贺菌中活性CRISPR/Cas系统与毒力基因的分布有关。     

Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens

Enterococci are now frequent causative agents of nosocomial infections. In this study, we analyzed the frequency and distribution of antibiotic resistance and virulence genotypes of Enterococcus isolates from broiler chickens. Fecal and cecal samples from nine commercial poultry farms were collected to quantify total enterococci. Sixty-nine presumptive enterococci were isolated and identified by API 20 Strep, and their susceptibilities to antibiotics were determined. Genotypes were assessed through the use of a novel DNA microarray carrying 70 taxonomic, 17 virulence, and 174 antibiotic resistance gene probes. Total enterococcal counts were different from farm to farm and between sample sources (P < 0.01). Fifty-one (74%) of the isolates were identified as E. faecium, whereas nine (13%), seven (10%), and two (3%) isolates were identified as E. hirae, E. faecalis, and E. gallinarum, respectively. Multiple-antibiotic resistance was evident in E. faecium and E. faecalis isolates. The most common multiple-antibiotic resistance phenotype was Bac Ery Tyl Lin Str Gen Tet Cip. Genes conferring resistance to aminoglycoside (aac, aacA-aphD, aadB, aphA, sat4), macrolide (ermA, ermB, ermAM, msrC), tetracycline (tetL, tetM, tetO), streptogramin (satG_vatE8), bacitracin (bcrR), and lincosamide (linB) antibiotics were detected in corresponding phenotypes. A range of 9 to 12 different virulence genes was found in E. faecalis, including ace, agg, agrB(Efs) (agrB gene of E. faecalis), cad1, the cAM373 and cCF10 genes, cob, cpd1, cylAB, efaA(Efs), and gelE. All seven E. faecalis isolates were found to carry the gelE gene and to hydrolize gelatin and bile salts. Results from this study showed the presence of enterococci of public and environmental health concerns in broiler chicken farms and demonstrated the utility of a microarray to quickly and reliably analyze resistance and virulence genotypes of Enterococcus spp.     

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification

AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.     

Isolation of Aeromonas hydrophila from a metropolitan water supply: seasonal correlation with clinical isolates.

The occurrence of Aeromonas spp. in the metropolitan water supply of Perth, Western Australia, Australia, was monitored at several sampling points during a period of 1 year. Water within the distribution system conformed to international standards for drinking water but contained Aeromonas spp. in numbers comparable to those in raw surface water, although this water was free of Escherichia coli. Coliforms and E. coli were found in raw surface waters, and Aeromonas spp. were found in raw water from surface and underground sources. Chemical treatment, followed by chlorination at service reservoirs, resulted in water free of E. coli and a decrease in the number of Aeromonas spp. Aeromonas spp. were found in the greatest numbers in summer. Multiple regression analysis showed that growth of Aeromonas spp. in chlorinated water was related to water temperature, residual chlorine, and interaction between these variables. The incidence of Aeromonas-associated gastroenteritis, determined from isolates referred to us for enterotoxin testing, paralleled the pattern of isolation of Aeromonas spp. in water within the distribution systems. We suggest that the presence of Aeromonas spp. in drinking water needs public health appraisal and that further work should be undertaken to permit reevaluation of standards for the quality of drinking water.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.