Antimicrobial resistance of Listeria spp. recovered from processed bison

AIMS: The current study examined the antimicrobial susceptibility of 86 Listeria spp. isolated from processed bison carcasses. MATERIALS AND METhods: Susceptibility to 25 antimicrobial agents was determined using E-test and National Antimicrobial Resistance Monitoring System (NARMS) panels. Most Listeria isolates (88-98%) exhibited resistance to bacitracin, oxacillin, cefotaxime, and fosfomycin. Resistance to tetracycline (18.6%) was also common. Of the 16 tetracycline-resistant Listeria isolates, 15 carried tetM and 2 contained integrase of Tn1545 transposons. Rifampicin and trimethoprim-sulfamethoxazole were the most active antimicrobial agents against Listeria spp., with a MIC(90) of 0.38 microg ml(-1). Ampicillin, erythromycin, penicillin, gentamicin, and tobramycin also exhibited good activity against Listeria spp., with MIC(90) not exceeding 1 microg ml(-1). Differences in resistance among Listeria spp. was displayed, as Listeria innocua strains were more resistant than other Listeria species. CONCLUSIONS: The study showed that Listeria monocytogenes strains from bison were susceptible to the antibiotics most commonly used to treat human listeriosis. However, the presence of antimicrobial resistance in L. innocua indicates the potential for transfer of resistance and a conjugative transposon to L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of our study will provide useful information for the development of public health policy in the use of antimicrobials in food animal production.     

单核增生李斯特菌Internalin A的克隆表达与抗体制备

目的克隆表达单核增生李斯特菌Internalin A（InlA）,并以之为抗原制备检测单核增生李斯特菌的抗体。方法通过PCR技术从单核细胞增生李斯特菌4b中扩增出inlA基因,克隆筛选和测序鉴定后,最终构建该基因的原核表达质粒pGEX-4T-InlA,谷胱甘肽树脂亲和层析纯化表达产物后,免疫小鼠分别制备相应的多抗和单抗。结果在大肠埃希菌中成功表达了InlA,并对其进行了纯化,融合表达产物分子量约为110 kD;免疫小鼠获得的抗血清效价达到1∶1600;得到了3株抗InlA的单克隆抗体杂交瘤细胞株,腹水单抗效价为1∶1×10^5-1∶3×10^5。2种抗体与其他病原菌均无交叉反应。结论通过表达单核增生李斯特菌的特异性蛋白制备的抗体,能有效地消除交叉反应,提高检测的特异性。     

新生儿及产妇感染单核细胞增生李斯特菌分离株的血清型、耐药性及分子特征研究

为探讨广西南宁地区新生儿及产妇感染的单核细胞增生李斯特菌(Listeria monocytogenes, Lm)的血清型、药物敏感性及其分子流行病学特征,本研究回顾性收集2015-2017年广西壮族自治区妇幼保健院新生儿科及产科送检标本中分离的Lm,对其进行体外药物敏感性检测、血清学分型以及多位点序列分型(multilocus sequence typing, MLST)分析菌株间的同源性;同时分析患儿及其母亲的临床特征及危险因素。结果显示,广西南宁地区新生儿感染Lm发病率较低,2015-2017年发病率为0.091‰;所有分离的Lm分属4b(83.3%)和1/2a(16.7%)2个血清型;药物敏感性试验结果显示,Lm对青霉素、氨苄西林、复方新诺明及美罗培南均100%敏感,暂未发现耐药菌株;MLST分型共获得2个序列型(sequence types,ST),以ST­1型(83.3%)为主。其中分离自同一新生儿患者(Case 2)外周血(Lm2)、耳拭子(Lm3)及其母亲羊水(Lm4)、宫颈分泌物(Lm5)的4株菌具有相同的血清型、药物敏感性表型以及MLST分型。感染Lm的患儿主要表现为发热、肺炎、发绀、败血症及脑膜炎;而产妇感染则具有非特异性的临床特征。结果提示,广西南宁地区存在的Lm菌株为致病性较强的4b、1/2a血清型菌株;Lm可通过母婴垂直传播引起新生儿感染。因此,临床医师应重视孕产妇及新生儿Lm病原学检查、早期诊断和及时合理地使用抗生素预防、治疗,从而减少Lm引起的母婴感染。     

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

AIM: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence-associated genes. METHODS AND RESULTS: A total of 305 samples comprising blood, urine, placental bits, faecal and vaginal swabs were collected from 61 patients with spontaneous abortions. Listeria spp. were isolated from 10 samples collected from nine (14.8%) patients. Confirmation of these isolates was based on biochemical tests, haemolysis on blood agar, CAMP test, phosphatidylinositol-specific phospholipase C (PI-PLC) assay followed by in vivo pathogenicity tests and multiplex PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap). Three isolates were confirmed as L. monocytogenes. Of these, two isolates turned out to be pathogenic and found to posses all five genes. However, the remaining two haemolytic L. monocytogenes isolates lacking the plcA gene and activity in the PI-PLC assay were found to be nonpathogenic by in vivo tests. CONCLUSIONS: The occurrence of pathogenic L. monocytogenes in cases of spontaneous abortions was 3.3%. It seems that the plcA gene and its expression have an important role as essential virulence determinants in pathogenic Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery of pathogenic L. monocytogenes isolates from cases of spontaneous abortion indicates the significance of listeric infection in pregnant women.     

李斯特菌溶血素基因的原核表达及其生物学特性

李斯特菌溶血素(LLO)是产单核细胞李斯特菌的主要毒力因子,利用PCR技术从血清型4b的产单核细胞李斯特菌菌株中扩增出编码LLO的hly基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达质粒pGEX6P1hly,SDSPAGE结果表明：LLO与谷胱甘肽在大肠杆菌中已融合表达,融合蛋白的分子量为82kD;溶血实验证明融合蛋白具有较强的裂解真核细胞膜的作用,表明表达产物LLO具有生物活性,其溶血效价达2.26×101.4 HU/mg,这为进一步研究其致病与免疫机理、单抗研制和疫苗设计提供了条件。     

Listeriosis in Sheep

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in healthy sheep. Acta vet. scand. 1979, 20, 168–179. — The excretion of Listeria monocytogenes (Lm) in the faeces and milk, and humoral and cell mediated immunity against Lm, were examined in a sheep flock where no cases of listeriosis had occurred during the last 3 years. The investigation was carried out during the indoor season. During the first part of the season 2 of the 10 pregnant, 8 months old lambs excreted Lm in the faeces, but none of the 106 ewes, 2–10 years old. At lambing the organism was isolated from the faeces of 6 of the 10 1 year old lambs and from 64% of the ewes, and from the milk of 1 of the lambs and 41% of the ewes. Nearly all the isolates (98.5%) belonged to serotype 1. Antibody titres against Lm were found in sera and whey by an indirect haemagglutination method. The titres were higher for the ewes than for the hoggs and seemed to be influenced by the number of foetuses the animals carried. Cell mediated immunity was determined by a skin test where delayed hypersensitivity against an antigen prepared from Lm, was measured. Animals fed grass silage had a stronger reaction than animals fed hay, and a stronger reaction was found in animals with ≥ 3 foetuses than in the remainder. The investigation indicates that even in a healthy sheep flock all the animals may be exposed to Lm, and the majority may be latent carriers and excrete this organism in the faeces and milk during periods of stress.     

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

Listeriosis at Vancouver General Hospital, 1965-79.

The records were reviewed of all patients treated at the Vancouver General Hospital over the 15 years from 1965 through 1979 for infections proved by culture to have been caused by Listeria monocytogenes. Although listeriosis is not common in humans, certain groups seem to be susceptible - immunocompromised patients, pregnant women, neonates and the elderly. All these groups were represented among the 22 cases reviewed. There were 17 adults, 3 of whom were pregnant women who had only a mild influenza-like illness. Of the remaining 14 adults 9 were immunocompromised and 5 apparently immunocompetent; 7 presented with meningitis and 7 with bacteremia only. Of the five infants with neonatal listeriosis, two had early-onset disease (bacteremia) and three had the late-onset form (meningitis). Seven patients were treated with penicillin alone, seven with ampicillin alone and eight with penicillin or ampicillin combined with kanamycin, gentamicin or chloramphenicol. There were eight deaths: several were directly attributable to the listeriosis, but in others the severity of the underlying illness was an important factor. Serotypes 1 and 4b were equally common among the 16 specimens of L. monocytogenes that were typed.     

A serologic study of canine herpes virus-1 infection in the Norwegian adult dog population

Canine herpes virus-1 (CHV1) causes a fatal hemorrhagic disease in neonatal puppies and is associated with reproductive problems in female dogs. This serologic study was conducted to assess the seroprevalence of CHV1 infection in Norway. Blood samples were collected from clinically healthy dogs (n = 436) one yr of age and older of both genders, supplied by four small animal clinics (A, B, C and D) in different parts of the country. The immunoperoxidase monolayer assay was used for testing of CHV1 antibodies. Serum titers were recorded as the reciprocal value of the highest dilution producing specific cell staining. Titers equal to or above 80 were considered positive for exposure to CHV1. In total, 80.0% of the dogs had titers ≥80 and were classified as positive. Mean age for seronegative dogs was 4.7 yrs (95% CI 4.1-5.4) and for seropositive dogs 5.0 yrs (95% CI 4.7-5.4). Of the dogs, 32.8% displayed a weakly positive titer of 80, whereas 41.5 and 5.7% fell into the moderately (titer 160 and 320) and strongly (titer ≥640) positive categories, respectively. No association was demonstrated when comparing CHV1 antibody titers to gender or reproductive parameters like previous matings, pregnancies, births or number of puppies born. Age, visit in foreign countries and clinic explained together 78% of the variation in antibody titer categories. The percentage of positive samples differed significantly between the four clinics (A 98%, B 58.5%, C 74.6%, D 89.5%). A reasonable explanation for this finding has not been established. No information about an ongoing outbreak of CHV1 infection was available. In conclusion, this study strongly indicates that CHV1 infection is endemic in the dog population of Norway. There are significant differences in seroprevalence between geographic regions in the country.     

The risk of Listeria monocytogenes infection in beef cattle operations

Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow-calf and feedlot) in central and southern California.
Methods and Results:  A repeated cross-sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf-cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut-grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots.
Conclusions:  Listeria monocytogenes was present at a higher proportion among cow-calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations.
Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow-calf operations.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Growth condition-related response of Listeria monocytogenes 412 to bacteriocin inactivation

Bacteriocin inactivation of Listeria monocytogenes 412 was studied as a function of growth phase. Cells were treated with nisin (300 IU ml-1) or pediocin (320 or 2560 AU ml-1) for 20 min at 30 degrees C. Inactivation with nisin or the low concentration of pediocin was growth phase dependent, with exponentially growing cells being more susceptible than stationary cells. No effect of growth phase was observed for the high pediocin concentration. Pediocin inactivation (320 AU ml-1) of L. monocytogenes 412 exposed to osmotic (6.5% NaCl) or low-temperature (5 degrees C) stress was investigated. Pediocin failed to inactivate osmotically stressed cultures and was unable to inhibit cold-stressed cells to the same degree as unstressed cells.     

RAPID DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

The aim of this study was to determine the prevalence of Listeria monocytogenes in packaged fresh ground turkey in Turkey using immunomagnetic separation (IMS) as a selective enrichment step in method and polymerase chain reaction (PCR). A total of 180 ground turkey samples were collected during a 1-year period. Thirty-two (17.7%) of the samples contained L. monocytogenes, 24 (13.3%) contained Listeria innocua, 7 (3.8%) had Listeria ivanovii and 5 (2.7%) had Listeria seeligeri by means of IMS-based cultivation method. A PCR assay was performed, based on hlyA gene-specific primers. In all L. monocytogenes isolates, hlyA gene was confirmed, indicating that the correlation between IMS-based cultivation and PCR methods was 100%. The results suggest that the prevalence of L. monocytogenes in ground turkey is relatively high in Turkey and that ground turkey should be produced under appropriate hygienic and technological conditions for the prevention of public health hazards.

PRACTICAL APPLICATIONS

Using fast and reliable methods to detect and identify foodborne pathogenic bacteria, including Listeria monocytogenes , is important to detect the risk of contaminated product and protect public health. In some ways it is time-consuming to isolate and identify the pathogenic microorganisms from food products using conventional techniques. Different methods or techniques can be used both for redounding the isolation chance and to gain time for this purpose. Immunomagnetic separation (IMS) and polymerase chain reaction (PCR) techniques are effective and rapid methods for separation, detection and confirmation of Listeria spp. from foods. In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates.     

单增李斯特菌prsA1基因的截短表达、纯化及多克隆抗体的制备

单增李斯特菌（Listeria monocytogenes）是一种常见的食源性致病菌,能够引发李斯特菌病,对食品安全构成巨大威胁。prsA1具有保守性和特异性,利用SignalP 4.1 Server程序、TMHMM Server V.2.0程序和SEPPA 2.0程序预测了PrsA1的信号肽段、跨膜区域及空间抗原表位,预测结果显示PrsA1的N端含有信号肽段及跨膜区且该蛋白具有良好抗原表位结构,因而可作为检测靶标。在此基础上,采用PCR法获得prsA1的非跨膜区序列即Δ84prsA1,构建重组质粒pET30a-Δ84prsA1并转入到大肠杆菌中诱导表达Δ28PrsA1,Ni-IDA柱亲和纯化重组蛋白Δ28PrsA1,以纯化的Δ28PrsA1为抗原制备多克隆抗体。间接ELISA检测多克隆抗体的效价,高达1∶128 000。Western blotting分析结果显示该多克隆抗体能够识别从单增李斯特菌中提取的PrsA1蛋白。利用生物信息学筛选检测靶标并分析抗原表位结构,最后成功制备了多克隆抗体,为单增李斯特菌检测靶标的筛选和免疫学检测提供了实践基础。     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

产单核细胞李斯特菌actA基因在大肠杆菌中的表达及其单克隆抗体的研制

利用PCR技术从血清型1/2a的产单核细胞李斯特菌Lm-4株中扩增出actA基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达载体pGEX-6P-1-actA及pET-actA,转入E·coli后,IPTG诱导目的蛋白的表达。SDS-PAGE结果表明,actA基因在两种载体中均获得表达,融合蛋白的大小分别约为120kDa和97kDa。以纯化蛋白为材料进行了ActA单抗的研制,获得4株抗ActA的单克隆抗体杂交瘤细胞株,腹水单抗ELISA效价为1∶5×104~1∶1×105。选取单抗1A5进行Westernblot分析,结果表明单抗1A5能和表达产物进行特异性反应,且与Lm-4多抗血清的Westernblot结果一致。actA基因的原核表达及单抗的研制为研究ActA蛋白的生物学活性及其致病作用奠定了基础。     

Survival and biofilm formation of Listeria monocytogenes in simulated vaginal fluid: influence of pH and strain origin

Listeria monocytogenes, the agent responsible for listeriosis, can be transmitted from mother to fetus/neonates by vertical transmission, transplacentally or during passage through the birth canal. The purpose of this study was to investigate the survival and biofilm formation of L. monocytogenes (isolated from clinical cases or from food) in simulated vaginal fluid at different pH values (4.2, 5.5 and 6.5). The results demonstrated that this pathogen is inhibited by the normal vaginal pH, but may proliferate when it increases. Clinical strains were significantly more resistant to pH 4.2 than food isolates. Listeria monocytogenes survived and even grew at the higher pHs investigated, suggesting that fetus/neonates from women having increased vaginal pH values during pregnancy may be at a higher risk of listeriosis. All isolates tested were producers of biofilm at different pH values; however, L. monocytogenes produced higher quantities of biofilm in a nutrient-rich medium. No significant differences in biofilm production were detected between food and clinical isolates. As L. monocytogenes are biofilm producers, this increases the probability of occurrence of neonatal infection.     

Role of endometrial blood flow assessment with color Doppler energy in predicting pregnancy outcome of IVF-ET cycles

This is a prospective study of 182 women (38 yrs or younger) undergoing IVF-ET. Endometrial thickness, echo pattern and blood flow on transvaginal ultrasonography were recorded eight hours prior to hCG administration. The patients were divided into three groups: A (n = 10) with undetectable endometrial blood flow; B (n = 82) with sub-endometrial blood flow; C (n = 90) with both endometrial and sub-endometrial blood flow. According to IVF-ET outcomes, all patients were re-divided into three groups: 1 non-pregnancy (n = 92); 2 intrauterine pregnancy with live fetus (n = 70); 3 others (n = 20 including biochemical pregnancy, embryonic diapause, ectopic pregnancy and miscarriage). Intrauterine pregnancy with live fetus in Group C (62.2%) was much higher than that in Group A and B (0% and 17.1%, p less than or equal to 0.001). The implantation rate (33.2%) was much higher than that in Group A and B (0% and 19.90%, p less than or equal to 0.001). The pulsatility index, resistance index, and S/D of endometrial spiral arteries were 0.1 +/- 0.2, 0.6 +/- 0.1 and 2.5 +/- 0.4 in Group 2, which were much lower than those in Group 1 and Group 3 (p1-2 less than 0.001, p2-3 less than 0.05). The patients with detectable endometrial blood flow had higher clinical pregnancy rates and implantation rates.     

DNA sequence heterogeneity in the gene encoding a 60-kilodalton extracellular protein of Listeria monocytogenes and other Listeria species.

Chromosomal DNA sequences from the 60 kilodalton protein gene of Listeria monocytogenes, amplified by the polymerase chain reaction, were used for restriction fragment length polymorphism differentiation of L. monocytogenes serotypes and other Listeria species. All 24 strains of L. monocytogenes examined produced an extracellular protein of molecular weight 60,000 (p60) as determined by Western blot analysis. Four of six other Listeria species had a protein that cross-reacted to antibodies to p60, but all differed in molecular weight, ranging from approximately 50,000 to 65,000. The gene encoding p60 was amplified from chromosomal DNA in all strains using polymerase chain reaction with a single primer pair. Restriction enzyme digestion with HindIII of the amplified product revealed a restriction pattern that was distinct between serotypes 1/2a and either 4b or 1/2b of L. monocytogenes. Of the other Listeria species, four strains that produced a cross-reacting protein likewise produced a polymerase chain reaction amplification product with the primer pair. Listeria innocua alone had a restriction pattern similar to that of Listeria monocytogenes serotype 4b and 1/2b. Genotypic heterogeneity, as revealed by DNA amplification and restriction endonuclease digestion of the p60 open reading frame, correlates with "electrophoretic type" grouping and may be related to differences in virulence mechanisms of Listeria monocytogenes and other Listeria species.