猪肉中单核细胞增生李斯特菌的分离与耐药性研究

了解猪肉中单核细胞增生李斯特菌的污染情况、血清型和耐药性现状。按照国标GB 4789.30-2010程序分离猪肉中单核细胞增生李斯特菌;多重PCR确定血清型;纸片扩散法检测单核细胞增生李斯特菌对抗生素的敏感性。研究结果表明273份猪肉样本中有52（19.02%)个受单核细胞增生李斯特菌的污染,共检出78株该致病菌。33株（42.31%）、24株（30.71%）、16株（20.51%）、3株（3.85%）、2株（2.56%)分别被确定为血清型1/2a（或3a）、1/2b（或3b或7）、1/2c（或3c）、4a（或4c）和4b（或4d或4e）。所有菌株对氨苄西林、 氨苄西林/舒巴坦、亚胺培能、环丙沙星、左氧氟沙星、万古霉素都敏感,对四环素（20.51%）的耐药率最为严重。11.54%的菌株对临床治疗李斯特菌病的青霉素、庆大霉素、红霉素等抗生素出现耐药。tet(M)基因是单核细胞增生李斯特菌耐四环素的主要机制之一,接合型转座子Tn916与该菌四环素耐药散播有直接关系。     

加工环境中单核细胞增生性李斯特菌耐药特征分析

目的分析在流通过程中加工环境来源的食源性李斯特菌耐药性及耐药基因携带情况,探讨加工环境对该菌的影响。方法收集2016~2017年分离自加工环境中的71株单核细胞增生性李斯特菌,利用微量肉汤稀释法对8种抗菌药物进行药物敏感性分析,并采用PCR鉴定11种耐药基因(tet A、tet M、tet S、erm A、erm B、erm C、mec A、aac(6’)-Ib、van A、van B、cfr)。结果 71株Lm对庆大霉素(11.27%)、氨苄西林(4.22%)、头孢他啶(98.59%)、环丙沙星(97.18%)、四环素(21.12%)、红霉素(15.49%)、林可霉素(92.96%)均具有不同程度的耐药性,对万古霉素敏感。其中以耐受3~6种抗菌药为主,耐药情况较为严重。耐药基因检测表明四环素类tet A,tet M为主要耐药基因,检出率高达50%以上,其次为红霉素类erm A、erm B、erm C,检出率约11%,氨基糖苷类aac(6’)-Ib的检出率为5.63%。结论加工环境单核细胞增生性李斯特菌的耐药性呈上升趋势,其耐药表型与耐药基因型并不完全一致,从而表明加工环境中存在影响单核细胞增生性李斯特菌耐药性因素,同时为食源性李斯特菌病的防治及预警提供参考依据。     

台州市食源性单核细胞增生李斯特菌分子特征研究

目的了解台州市食品中分离的单核细胞增生李斯特菌的血清型、毒力基因以及基因分型情况,建立食源性单核细胞增生李斯特菌的分子特征本底信息,为食源性疾病的防治提供技术支持。方法对近几年从食品中分离的37株单核细胞增生李斯特菌进行多重PCR血清分型、9种毒力基因(prf A、inl A、inl B、iap、fla A、hly A、plc B、mpl和act A)PCR检测、PFGE基因分型,用Bio Numerics 6.6软件对分型数据进行聚类分析。结果 37株食源性单核细胞增生李斯特菌的血清型以1/2a或3a型别为主;所有菌株均检出4种以上毒力基因,有15株菌携带所有9种毒力基因;37株菌经Apa I酶切PFGE分型后,共得到22种带型,每种带型包含1~5株不等,相似度区间为67%~100%。结论台州市食源性单核细胞增生李斯特菌存在致病流行风险,建立的指纹图谱数据库可为食源性疾病的防治提供技术支持。     

一株食源性多重耐药单核细胞增生李斯特菌的耐药性研究

本研究对食品样本中分离的一株多重耐药单核细胞增生李斯特菌进行耐药机制的探讨,以期对食源性单核细胞增生李斯特菌多重耐药现象的控制提供理论依据。本文通过聚合酶链式反应筛选耐药决定因子,质粒消除及自然转化实验对耐药决定因子进行定位及传播能力的探讨,最后通过传代实验验证该菌株多重耐药性传播的稳定性。结果表明,对检测到的多重耐药菌株LM78(耐受氯霉素、红霉素、链霉素、四环素、复方新诺明)进行相关耐药基因检测,检测到cat、erm B、tet S 3个耐药基因。质粒消除后MIC值下降到敏感范围,且该质粒可通过自然转化在不同菌属间传递,说明这些耐药基因存在于质粒上。该质粒在无抗生素选择压力下连续传代,仍具有较高稳定性。食源性致病菌多重耐药性有可能通过不同细菌种属间转移,进而由食物链向人类传播,对人类健康造成潜在的威胁。     

单核细胞增生李斯特菌的毒力基因检测及PFGE分型的研究

目的了解福建省食品中单核细胞增生李斯特菌携带hly、plcA、plcB和prfA毒力基因的情况及脉冲场凝胶电泳(PFGE)的分型情况。方法将hly、plcA、plcB和prfA基因作为靶序列选取4对引物,通过聚合酶链反应(PCR)检测61株单核细胞增生李斯特菌和2株可疑单核细胞增生李斯特菌的毒力基因,用PulseNet单核细胞增生李斯特菌标准方法进行7株单核细胞增生李斯特菌的PFGE分子分型。结果61株单核细胞增生李斯特菌毒力基因为hly 、plcA 、plcB 和prfA ,2株可疑单核细胞增生李斯特菌的毒力基因分别为hly-、plcA 、plcB-、prfA-和hly-、plcA-、plcB-、prfA-。7株单核细胞增生李斯特菌的PFGE分为5个型。结论实验结果表明福建省食品中分离到的单核细胞增生李斯特菌均含有hly、plcA、plcB和prfA基因,属于致病株。对2株可疑单核细胞增生李斯特菌进行了进一步的鉴定,排除了单核细胞增生李斯特菌,该毒力基因检测方法可用于可疑单核细胞增生李斯特菌的进一步鉴别。7株菌中有两对2株PFGE型别一致,一致的菌株来自不同年份不同销售地点的同一品牌,应用PFGE方法可以进一步调查该厂冻鸡肉中单核细胞增生李斯特菌的传播途径和污染源。     

北京市人源性单核细胞增生李斯特菌耐药特征及分子分型研究

目的研究北京市人源性单核细胞增生李斯特菌的血清学分型、耐药及分子特征。方法对2013—2015年北京市李斯特菌病专项监测中分离的32株单核细胞增生李斯特菌进行PCR法血清学分型和PFGE分型,并采用CLSI和EUCAST推荐的微量肉汤稀释法检测菌株对12种抗生素的敏感性。结果 32株单核细胞增生李斯特菌共分为4种血清型,其中位居前2位的血清型是1/2b和1/2a,分别为17株和12株,4b血清型2株,1/2c血清型1株。32株菌经Asc I酶切共分为23种PFGE带型,分为4个簇,相同血清型李斯特菌的PFGE带型聚集成簇,两种分型方法一致性为93.75%(30/32)。32株单核细胞增生李斯特菌均对青霉素、氨苄西林、复方新诺明及红霉素敏感,大部分菌株对其他8种抗生素的MIC值较小,但有少数菌株对苯唑西林和四环素的MIC值较大,高达32、64μg/ml。结论北京市人源性单核细胞增生李斯特菌血清型以1/2b和1/2a为主,PFGE图谱与血清学分型存在一定相关性,耐药率普遍较低,但需加强耐药趋势监测。     

1例急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病原学及分子分型特征分析

目的对北京市1名急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病例进行病因溯源,对分离到的单核细胞增生李斯特菌进行血清学分型、耐药及分子分型研究。方法对患者不同时期外周血分离的2株单核细胞增生李斯特菌和1株环境涂抹样品单核细胞增生李斯特菌分离株进行血清学分型、耐药性分析、脉冲场凝胶电泳(PFGE)和多位点序列分析(MLST)。结果 3株单核细胞增生李斯特菌均为1/2a-3a血清型,耐药结果一致,均对青霉素、氨苄西林、复方新诺明、美罗培南及红霉素敏感,3株菌的PFGE带型一致,MLST型别均为ST155。结论本研究中患者生活环境中存在单核细胞增生李斯特菌的污染情况,高度怀疑患者感染单核细胞增生李斯特菌与其生活环境中分离到的菌株为同一来源。     

中国食源性单核细胞增生李斯特菌耐药特征分析

目的研究中国22个省市自治区9类食品中分离的单核细胞增生李斯特菌的耐药特征。方法采用微量肉汤稀释法测定了1 069株单核细胞增生李斯特菌对庆大霉素、氨苄西林、青霉素、四环素、强力霉素、亚胺培南、红霉素、环丙沙星、左旋氧氟沙星、头孢噻吩、利福平、万古霉素、氯霉素、氨苄西林/舒巴坦酸和复方新诺明15种抗生素的耐药性。结果 1 069株单核细胞增生李斯特菌耐药率为6.92%,主要耐受的抗生素有四环素、强力霉素、红霉素、氯霉素和环丙沙星,并出现了多重耐药株。在不同食品来源中,分离自速冻米面制品的菌株耐药率最高,为9.64%。在22个省市自治区中,耐药率前3位的是:甘肃、吉林、福建,分别为27.3%、20.4%和17.4%。结论中国食源性单核细胞增生李斯特菌的耐药率较低,不同地区、不同食物来源耐药率差异较大,仍要加强对生产和临床使用抗生素的管理,减缓单核细胞增生李斯特菌耐药趋势的上升。     

云南省牛乳中单核细胞增生李斯特氏菌的分布特征、耐药性及毒力研究

目的分析云南省牛乳中单核细胞增生李斯特氏菌(Listeria monocytogenes)的分布特征、耐药性及毒力现状。方法采集云南省主要奶产区牛乳样本,按照GB 4789.30—2016方法分离单核细胞增生李斯特氏菌疑似菌株,采用飞行时间质谱技术结合16SrDNA测序对疑似菌株进行鉴定;采用微量肉汤稀释法对分离菌株进行抗生素药敏实验;通过聚合酶链式反应(polymerasechainreaction,PCR)技术检测单核细胞增生李斯特氏菌中7种毒力基因的存在状况。结果158份样品共有4份检出单核细胞增生李斯特氏菌,检出率为2.53%;共分离获得8株菌。其中,生鲜乳中检出率为3.36%,巴氏杀菌乳中未检出。在生鲜乳中,荷斯坦牛乳、水牛乳、牦牛乳检出率分别为2.13%、0.00%和20.00%;标准化牛场、奶牛合作社和个体户的检出率分别为2.50%、1.51%和15.38%;手工挤奶和机械挤奶检出率为11.76%和1.96%。药敏实验结果显示8株菌均对青霉素耐药,对四环素、复方新诺明、红霉素耐药率分别为87.50%、75.00%和50.00%;多重耐药率为75.00%;但所有菌株对氨苄西林均敏感。8株菌对7种毒力基因的携带程度从12.50%~75.00%不等。结论云南省牛乳尤其生鲜乳中存在一定程度的单核细胞增生李斯特氏菌污染,且该菌具有普遍耐药性并携带一定程度的毒力基因,对消费者的安全具有潜在风险,应加强卫生监督管理。     

2012—2015年北京市西城区单核细胞增生李斯特菌多位点序列分型及耐药研究

探讨单核细胞增生李斯特菌多位点序列分型(MLST)与耐药性的关联性,确定某些具有高致病性潜能的流行克隆株的存在。方法 采用Kirby-Bauer(K-B)纸片扩散法和E-test药敏试条法对14种抗生素进行药物敏感性试验,以MLST技术对50株菌株进行基因分型。结果 单核细胞增生李斯特菌耐药率为22.00%(11/50),并出现多重耐药株。50株单核细胞增生李斯特菌MLST分析共获得12个型别,以ST9和ST121为优势型别。结论 特定ST型别在食品生产过程中存在特定菌株之间的传递,人源性和食源性单核细胞增生李斯特菌中均发现耐药株,可能存在耐药基因的传递,应加强对具有潜在致病性的ST型别的监测力度。     

Identification of tet(M) in two Lactococcus lactis strains isolated from a Spanish traditional starter-free cheese made of raw milk and conjugative transfer of tetracycline resistance to lactococci and enterococci

Specific PCR and sequencing showed that a tet(M) gene was present in two tetracycline-resistant Lactococcus lactis strains isolated from a raw milk, starter-free cheese. Hybridisation experiments using as a probe an internal segment of the gene obtained by PCR associated tet(M) with plasmids of around the same size (30 kbp) in both strains. Molecular analysis of the tetracycline resistance loci, including the upstream and downstream regions of the genes, showed them to be identical to one other and to the tet(M) encoded by the conjugative transposon Tn916. Amplification of Tn916-derived segments suggested the transposon was complete in the two L. lactis strains. Further, curing of the tetracycline resistance was accompanied by a reduction in size of the plasmids comparable to that expected for Tn916. Tetracycline resistance could be transferred by conjugation to plasmid-free Lactococcus and Enterococcus strains. However, no plasmid DNA was detected among the transconjugants while both tet(M) and transposon-related sequences were amplified by PCR. This suggested that only the transposon was mobilized.     

Abundance and distribution of tetracycline resistance genes and mobile elements in an oxytetracycline production wastewater treatment system

We investigated the abundance and distribution of tetracycline resistance genes (tet genes) in an oxytetracycline (OTC) antibiotic production wastewater (APW) treatment system. Of sixteen tet genes and five mobile elements, nine tet genes (tet(A), tet(C), tet(G), tet(L), tet(M), tet(O), tet(Q), tet(W), and tet(X)) and two mobile elements (class 1 integron (intI1) and transposon Tn916/1545) were successfully quantified by real-time PCR. The relative abundance of tet genes in the effluent and activated sludge (1.2 × 10(-4) to 1.3 × 10(0)) of the APW treatment system were up to 2 orders of magnitude higher than those in the OTC fermentation residues (8.5 × 10(-5) to 6.7 × 10(-3)) (P < 0.01), and 1-4 orders of magnitude higher than those in sewage and nonantibiotic production wastewater treatment systems (P < 0.01), showing that the discharge of abundant tet genes from the APW treatment system is worthy of attention. The three most abundant genes in the APW treatment system, tet(A), tet(C), and tet(G), exhibited significant positive correlations with intI1 (R(2) = 0.73, 0.95, and 0.83, respectively; P < 0.05), suggesting that intI1 may be involved in their proliferation. This is the first study showing that some measures may be required to control the discharge of antibiotic resistance genes from treated APW and activated sludge.     

Antimicrobial susceptibilities of Listeria monocytogenes strains isolated from food and food processing environment in Poland

A total of 471 Listeria monocytogenes isolates from different types of food and food-related sources in Poland during 2004-2010 were examined. This number includes 200 isolates from fish, 144 from fresh and frozen vegetables, 43 ready-to-eat products (deli foods, cold cuts), 13 from dairy products, 16 from raw meats, 15 from confectionery products and 40 directly from processing plants. All isolates were subjected to serotyping and lineage assays using PCR, and antimicrobial susceptibility using E-test and a broth microdilution method. Of all isolates, 256 (54.4%), 120 (25.5%), 59 (12.5%), 36 (7.6%) were identified as serotypes 1/2a (or 3a), 1/2c (or 3c), 1/2b (or 3b or 7), and 4b (or 4d or 4e), respectively. A direct correlation between the most common serotypes and three L. monocytogenes lineages was also observed. All L. monocytogenes isolates belonged to lineages I (20.2%) and II (79.8%). All strains were sensitive to ampicillin, amoxicillin, gentamicin, erythromycin, trimethoprim, rifampicin, vancomycin, chloramphenicol and sulfamethoxazol. Two of the L. monocytogenes strains (0.42%) showed phenotypic resistance. One strain was resistant to tetracycline and minocycline due to the presence of tet(M). It did not carry gene int, which may indicate that the tet(M) gene in this strain was not integrated in the transposon Tn916-Tn1545 family. The resistance of the second strain to ciprofloxacin and norfloxacin was attributed to active efflux associated with overexpression of gene lde. Our data indicate the low prevalence of antimicrobial resistance among L. monocytogenes isolates from food and food-related sources in Poland.     

Indirect evidence of transposon-mediated selection of antibiotic resistance genes in aquatic systems at low-level oxytetracycline exposures

Subinhibitory levels of antibiotics can promote the development of antibiotic resistance in bacteria. However, it is unclear whether antibiotic concentrations released into aquatic systems exert adequate pressure to select populations with resistance traits. To examine this issue, 15 mesocosms containing pristine surface water were treated with oxytetracycline (OTC) for 56 days at five levels (0, 5, 20, 50, and 250 microg L(-1)), and six tetracycline-resistance genes (tet(B), tet(L), tet(M), ted(O), tet(Q), and tet(W)), the sum of those genes (tet(R)), "total" 16S-rRNA genes, and transposons (Tn916 and Tn 1545) were monitored using real-time PCR. Absolute water-column resistance-gene abundances did not change at any OTC exposure. However, an increase was observed in the ratio of tet(R) to 16S-rRNA genes in the 250 microg L(-1) OTC units, and an increase in the selection rate of Tc(r) genes (relative to 16S-rRNA genes) was seen when OTC levels were at 20 microg L(-1). Furthermore, tet(M) and Tn916/1545 gene abundances correlated among all treatments (r2 = 0.701, p = 0.05), and there were similar selection patterns of tetR and Tn916/1545 genes relative to the OTC level, suggesting a possible mechanism for retention of specific resistance genes within the systems.     

Antibiotic survey of Lactococcus lactis strains to six antibiotics by Etest, and establishment of new susceptibility-resistance cut-off values

In order to establish cut-off values for Lactococcus lactis to six antibiotics to distinguish susceptible and intrinsically resistant strains from those having acquired resistances, the minimum inhibitory concentration (MIC) of tetracycline, erythromycin, clindamycin, streptomycin, chloramphenicol and vancomycin was determined in 93 different Lc. lactis strains using the Etest. These bacterial strains were originally isolated from dairy and animal sources in widely separated geographical locations. Cut-offs were defined on the basis of the distribution of the MICs frequency of the studied antibiotics, which in the absence of acquired determinants should approach to a normal statistical distribution. In general, the new cut-off values proposed in this study are higher than previously defined (European Commission, 2005. The EFSA Journal 223, 1-12). Based on these new values, all the strains tested were susceptible to erythromycin, chloramphenicol and vancomycin, and 79 susceptible to all six antibiotics. However, 11 strains (around 12%) were considered resistant to tetracycline (six of which had been identified after screening of a large collection of lactococci strains for tetracycline resistance) and five (5.4%) resistant to streptomycin. Of these, two fish isolates proved to be resistance to both tetracycline and streptomycin. From the tetracycline resistant strains, tet(M) and mosaic tet(L/S) genes were amplified by PCR, demonstrating they harboured acquired antibiotic resistance determinants.     

Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products

Escherichia coli is used to assess the hygienic quality of food products and the dissemination of antimicrobial resistance. In particular, tetracycline-resistant E. coli can be chosen as an indicator of antibiotic resistant bacteria because it has a high frequency of occurrence. The purpose of this study was to investigate the distribution and transfer of tetracycline resistance determinants in meatborne E. coli. A total of 121 tetracycline-resistant E. coli isolates were collected from meat and meat products (raw meat, fish, and processed foods) from 2004 to 2006 in Korea. Among these isolates, tet(A) (52.4%) was the most frequent tetracycline resistance determinant, followed by tet(B) (41.3%), whereas tet(C) (1.7%) and tet(D) (0.8%) were less frequently identified. Two isolates (1.6%) contained two tet genes simultaneously, tet(A) and tet(B). Minimal inhibitory concentrations (MICs) to tetracycline family antibiotics, such as tetracycline, minocycline, doxycycline, oxytetracycline, and chlortetracycline were higher for isolates carrying the tet(B) gene compared to isolates carrying tet(A) (P<0.0001). Conjugation experiments were performed by the broth mating method; 119 isolates (98.3%) containing at least one of the tet genes were shown to transfer tetracycline resistance to recipient E. coli J53. Also, we observed high diversity of tetracycline-resistant E. coli isolates in meat and meat products in Korea by using XbaI pulsed-field gel electrophoresis (PFGE) typing. This study suggests that the high prevalence of tetracycline-resistant E. coli in meat may be due to the high transferability of tet determinants.     

Antibiotic susceptibility of Bifidobacterium thermophilum and Bifidobacterium pseudolongum isolates from animal sources

The widespread use of antimicrobial substances has led to resistant populations of microorganisms in several ecosystems. In animal husbandry, the application of antibiotics has contributed to resistance development in pathogenic and commensal bacteria. These strains or their resistance genes can be spread along several ecological routes, including the food chain. Antibiotic resistance is important in terms of the safety of industrial strains, such as probiotics for food and feed. Bifidobacterium thermophilum and Bifidobacterium pseudolongum are known to comprise the major part of the bifidobacterial microbiota in the gut and feces of cattle and pigs. In this study, the antimicrobial susceptibility in bifidobacterial isolates of these species was investigated. Isolates from the beef and pork production chain were identified and typed to strain level, and the antimicrobial susceptibility level was tested to a set of antibiotics. Isolates with low susceptibility levels were screened by PCR for already described resistance genes. Strains atypically resistant to clindamycin, erythromycin, and tetracycline were determined. The resistance genes tet(O), tet(W), and erm(X) were detected in the bifidobacterial species that were examined.     

Phenotyping and genotyping of antibiotic-resistant Escherichia coli isolated from a natural river basin

Scientists have become increasingly concerned about the occurrence of antibacterial resistance in the environment. In this study, Escherichia coli resistant to one or more antibiotics among nine antibiotics was screened from Wenyu River Basin in Beijing, China, with mean frequency of 48.7 +/- 8.7% of 388 isolates in summer and 47 +/- 6% of 236 isolates in winter. The mean multiantibiotic resistance (MAR) index in summer was 0.11 +/- 0.03, slightly lower than that (0.14 +/- 0.04) in winter. Most frequent resistance appeared for sulfonamides, tetracycline, and ampicillin. The distribution of 20 tetracycline, three sulfonamide, and three beta-lactam resistance genes was assessed in the resistant isolates. While 97% of the ampicillin (AMP) resistant mechanism could be explained by the resistance gene TEM, 90% of the tetracycline (TC) and 96% of the sulfonamide (SXT) resistances could be explained by tet(A), tet(B), tet(M), and their combinations and sul(I), sul(II), sul(III), and their combinations, respectively. tet(M), a tetracycline-resistant gene originally detected in Gram-positive bacteria, and its combinations with tet(A) or tet(B) were first detected in E. coli isolated from a natural river basin, suggesting that tet(M) in E. coli might have been transferred from other bacterial species through horizontal gene transfer, which was supported by the fact that no tet(M) was detected in the isolates of human and chicken sources, except for only one isolate from swine. The source of sulfonamide-resistant E. coli in the river was supposed to be mainly from humans, based on a comparison of the sulfonamide resistance genotypes in animals and humans.     

Vancomycin-susceptible dairy and clinical enterococcal isolates carry vanA and vanB genes

A total of 109 enterococcal isolates from dairy food products and from human and dog infections, isolated in Portugal, and 26 type and reference strains of the genus Enterococcus were screened for vancomycin resistance. MIC values, both for vancomycin and teicoplanin, were determined. The genetic relatedness of isolates carrying either vanA and/or vanB was determined using Pulsed Field Gel Electrophoresis. For vanA carrying isolates, transposon Tn1546 was partially mapped using PCR. None of the 59 dairy isolates was resistant to vancomycin. Among the 50 clinical isolates, only one, carrying vanB, behaved as resistant, with a MIC value of 256 microg/mL. The type and reference strains used were susceptible both to vancomycin and teicoplanin. vanA was found in 37% of the dairy isolates and 40% of the clinical isolates. vanB was only detected in 18% of the clinical, both human and dog, isolates. PCR partial mapping of Tn1546 revealed 23 different patterns among 42 isolates. Some patterns were shared between dairy and clinical isolates. Using Pulsed Field Gel Electrophoresis six groups of isolates were found to be genetically undistinguishable and grouping was found to be geographically and location specific/related. No genetic relatedness was found between isolates from dairy, human and veterinary sources. These results show that an incomplete and/or unfunctional Tn1546 element may explain the absence of resistant behaviour in the studied isolates, even when vanA gene is present. Moreover, the work reported shows that both clinical (human and animal) and dairy isolates have been in contact with VanA genotype of resistance and suggest that dissemination of vanA gene has been through transposable elements, like Tn1546, and not by clonal dissemination of a resistant strain. Therefore, a national strategy should be implemented to survey both vancomycin resistance and its genetic dissemination.