Cooperation between wild lactococcal strains for cheese aroma formation

Several wild lactococcal strains were tested for their ability to produce aroma compounds during growth in milk. Strains were incubated alone and in combination with Lactococcus lactis IFPL730, which is characterized by showing α-keto acid decarboxylase activity. Volatile compounds from incubated milks were analyzed by means of solid-phase microextraction (SPME) and evaluated by gas chromatography–mass spectrometry (GC–MS). Incubated milks were also sniffed for sensory analysis to describe aroma attributes. The combination of L. lactis IFPL326 that showed the highest branched chain aminotransferase activity with IFPL730 contributed to the highest formation of leucine-derived volatile compounds, such as 3-methylbutanal, 3-methyl-1-butanol and 2-hydroxy-4-methyl pentanoic acid methyl ester. In addition, the milk incubated with this combination of strains was awarded, by the test panellists, the highest scores for “ripened cheese” attribute and aroma intensity. The results indicate that combination of L. lactis strains harbouring complementary catabolic routes can contribute to improved cheese aroma formation, the combined cultures with L. lactis IFPL730 resulting in higher volatile compound formation than isolate strains.     

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMβ1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.     

Inhibition by Lactobacillus sakei of other species in the flora of vacuum packaged raw meats during prolonged storage

The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm⁻² of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.     

Lactococcal 936-species phage attachment to surface of Lactococcus lactis

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.     

Validation of a model for growth of Lactococcus lactis and Listeria innocua in a structured gel system: effect of monopotassium phosphate

The effect of monopotassium phosphate (KH(2)PO(4)) on the chemical environment and on growth of Listeria innocua and Lactococcus lactis in coculture were investigated in a liquid and in a gelled microbiological medium at 12 degrees C and an initial pH of 6.2. As expected, addition of KH(2)PO(4) to both the liquid and gelled media resulted in an increase in buffering capacity. This effect on buffering capacity changed the profiles of lactic acid dissociation and pH evolution. At all gelatin concentrations studied, addition of KH(2)PO(4) increased the growth rate and the stationary cell concentration of L. lactis. In addition, the growth rate of L. innocua slightly increased but, in contrast, the stationary cell concentration remained unchanged. A new class of predictive models developed previously in our research team to quantify the effect of food model gel structure on microbial growth [Antwi, M., Bernaerts, K., Van Impe, J. F., Geeraerd, A. H., 2007. Modelling the combined effect of food model system and lactic acid on L. innocua and L. lactis growth in mono- and coculture. International Journal of Food Microbiology 120, 71-84] was applied. Our analysis indicate that KH(2)PO(4) influenced the parameters of the chemical and microbiological subprocesses of the model. Nonetheless, the growth model satisfactorily predicted the stationary cell concentration when (i) the undissociated lactic acid concentrations at which L. innocua and L. lactis growth cease were chosen as previously reported, and (ii) all other parameters of the chemical and microbiological subprocesses were computed for each medium. This confirms that the undissociated lactic acid concentrations at which growth ceases is a unique property of a bacterium and does not, within our case study, depend on growth medium. The study indicates that microbial growth depends on the interplay between the individual food components which affect the physicochemical properties of the food, such as the buffering capacity. Towards future research, it can be concluded that mathematical models which embody the effect of buffering capacity are needed for accurate predictions of microbial growth in food systems.     

Technical note: Use of RFLP to characterize Lactococcus lactis strains producing exopolysaccharides

Restriction fragment length polymorphism (RFLP) is used to differentiate microorganisms by analysis of their DNA restriction patterns. A modified RFLP procedure is proposed for the rapid characterization of Lactococcus lactis strains producing exopolysaccharides (EPS). The availability of such effective cataloging system is likely to benefit research aimed at identifying lactococcal strains that produce novel EPS.     

基于全基因组测序技术的食品分离致泻大肠埃希菌的分子特征及耐药性研究

目的 了解上海市肉品和水产品中分离致泻大肠埃希菌（DEC）的生物学特征及耐药性,为防控由该菌引起的食源性疾病提供科学依据。方法对食品分离DEC菌株进行全基因组测序及药物敏感性试验,利用BioNumerics软件对全基因组测序数据进行拼接,利用拼接序列开展多位点序列分型、毒力基因和耐药基因分析。结果本研究中56株DEC菌株中肠道聚集性大肠埃希菌（EAEC）占比最高,达73.2%,且以astA基因为主（90.2%）。56株DEC分为37个ST型,显示高度多样性。DEC菌株对喹诺酮类抗生素耐药性较高（64.3%）,其次是对氨基糖苷类、β-内酰胺类抗生素和四环素类抗生素耐药,且多重耐药性菌株占48.2%,耐药基因相应的以喹诺酮类、氨基糖苷类、β-内酰胺类抗生素耐药基因为主,且大多数对抗生素耐药的菌株均携带其相应的耐药基因。结论 EAEC是上海市食品中分离DEC菌株的主要型别,对喹诺酮类、β-内酰胺类、氨基糖苷类抗生素的耐药率较高,且携带相应的耐药基因,全基因组测序技术可应用于DEC的分子生物学监测中,为疾病预防控制提供科学依据。     

Growth and metabolic behaviour of Lactococcus lactis subsp. lactis IPLA 947 in anaerobic lactose-limited chemostat cultures

Lactococcus lactis subsp. lactis IPLA 947 is a starter strain isolated from a Spanish farmhouse acid-coagulated cheese. To use this strain as starter culture in Afuegal Pitu cheese manufacture, anaerobic lactose-limited chemostat cultures were used to optimise the active biomass production in a simple medium (BRFS). Growth, lactose consumption and metabolite production were measured at different pH values (5.5, 6.0 and 6.5) and dilution rates (0.06 to 0.96 h^–1). The influence of both variables on the quoted parameters was described by means of mathematical models. These provided a satisfactory representation of the experimental data. The bacterial response surface showed a significant increase in the biomass when the pH increased and the dilution rate decreased. As dilution rate increased, biomass yield increased but only at the lowest pH. The lactose consumption and the ratio of metabolic end products were also influenced by both variables. As dilution rate decreased, the residual lactose decreased and increased amounts of metabolites other than lactate (acetate, formate, ethanol) were produced. The minimum lactose consumption was obtained at pH 5.5 and 0.96 h^–1 dilution rate. A significant rise of acetate and formate levels occurred as pH increased. A maximum lactate level, along with an acetate, formate and ethanol production close to the minimum, was predicted by mathematical modelling at 0.5173 h^–1 dilution rate and pH 6.054.

In vitro antimicrobial effect and in vivo preventive and therapeutic effects of partially purified lantibiotic lacticin NK34 against infection by Staphylococcus species isolated from bovine mastitis

Lantibiotics are small (<5 kDa), polycyclic peptides produced by gram-positive bacteria; they are also known as gram-positive bacteriocins. The high antimicrobial activity of lacticins and the continuing appearance of antibiotic-resistant bacteria in recent years have resulted in a renewed interest in lantibiotics. A partially purified form of lacticin NK34 (a Lactococcus lactis product isolated from the Korean fermented fish jeotgal) was tested to determine its antimicrobial effects against Staphylococcus aureus (n = 20) and coagulase-negative Staphylococcus (CNS, n = 20) strains isolated from the raw milk of cows with subclinical bovine mastitis in the present study. The spot-on-lawn assay was used to identify the 2 strains from each group with the greatest lacticin NK34 susceptibility, and the minimal lethal dose (MLD) was measured in ICR (imprinting control region) mice. The preventive and therapeutic effects of lacticin NK34 on the mouse infection model were determined for the first time. Lacticin NK34 demonstrated antimicrobial effects in 14 of 20 (70%) S. aureus indicator strains and in 18 of 20 (90%) CNS strains. Staphylococcus aureus 69 and S. simulans 55 demonstrated the greatest susceptibility to lacticin NK34 in the spot-on-lawn assay. The S. aureus 69 MLD was measured at 1.53 × 10⁹ cfu/mouse, whereas the S. simulans 55 MLD was 3.59 × 10⁹ cfu/mouse. Mice infected experimentally with S. aureus 69 MLD or S. simulans 55 MLD were treated with lacticin NK34. Treated mice demonstrated an 80% survival rate (48 of 60 mice) compared with a survival rate of 7.5% (3 of 40 mice) in control mice treated with distilled water. These data suggest that lacticin NK34 might be useful in the control of bovine mastitis and systemic bacterial infection.     

Analysis of hemin effect on lactate reduction in Lactococcus lactis

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.     

Analysis of antibiotic resistance patterns and detection of mecA gene in Staphylococcus aureus isolated from packaged hamburger

Presence of Staphylococcus aureus, antibiotic resistance pattern and PCR detection of mecA gene in isolated strains were investigated in total of 256 packaged hamburgers in Iran-Tehran. For this purpose we used standard disk-diffusion method and sensitive and specific PCR technique, respectively. Results showed that 25% of samples were positive for S. aureus. Resistance to meticillin, erythromycin, penicillin G, cefazolin, ciprofloxasin, vacomycin and amoxiclave was determined 89%, 20.3%, 18.7%, 15.6%, 14%, 26.6% and 12.5%, respectively. According to the obtained results from PCR analysis of methicillin-resistant S. aureus (MRSA), mecA gene was present in 100% of the resistant isolates, 0% of intermediate-resistance isolates and 25% of susceptible isolates. The results obtained from PCR detection of mecA gene showed high correlation with standard disk diffusion test.     

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx₂-positive, five of which were also stx₁-positive. The remaining isolate was positive only for the stx₁ gene. All stx-positive isolates (whether positive for stx₁, stx₂ or stx₁ and stx₂) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.     

Environmental factors for phage-induced fermentation problems: replication and adsorption of the Lactococcus lactis phage P008 as influenced by temperature and pH

The lactococcal phage P008 was investigated for its growth characteristics under certain environmental conditions. Phage growth was characterized by the latency period, the average burst size (determined in one-step growth experiments) and by the percentage of adsorption to the host cells after 1, 5, 10 and 15 min (determined in modified one-step growth experiments). The incubation conditions studied were temperatures from 20 to 40 degrees C and pH values of 4.8 and 6.4. The growth medium was ultrafiltration permeate of skim milk, which forms the water phase of milk. Both, the temperature and the pH influenced the growth parameters: Increased temperature as well as low pH led to a faster latency along with a higher average burst size. The percentage of adsorption was at maximum when the standard conditions of 30 degrees C and pH 6.4 were applied. Below pH 5, adsorption was reduced to below 10%. A decrease of temperature slightly reduced phage multiplication. Data were incorporated into a model to simulate phage multiplication under certain conditions, taking the percentage adsorption into account. The cell destruction of the host culture was calculated accordingly. The simulation (extrapolation) was validated by experimental growth curves of phages and phage-infected host cultures.     

Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses

Prevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3′IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

2017—2020年江苏省无锡市沙门菌的血清型与耐药性研究

目的 分析2017—2020年江苏省无锡市沙门菌报告病例的血清型特点和抗生素耐药性流行特征及变化趋势。方法 收集分离自 2017—2020年无锡市食源性疾病哨点监测医院腹泻病例（住院和门诊患者）标本中的216株沙门菌菌株,采用玻片凝集法对216株菌进行血清型鉴定及分析,并采用微量肉汤稀释法检测沙门菌对13种抗生素的药物敏感性。结果 216株沙门菌分为42种血清型,优势血清型分别为肠炎沙门菌28.70%（62/216）占比,鼠伤寒沙门菌26.39%（57/216）。耐药性分析结果显示,216株沙门菌对美洛培南、阿米卡星、阿奇霉素和头孢他啶高度敏感,高敏菌株占比均高于90%;所有沙门菌对氨苄西林的耐药率最高,达到68.06%,对美洛培南的耐药率最低,仅0.46%;沙门菌在2017—2020年对氨苄西林的耐药率呈逐年增高的趋势,且每年沙门菌分离株对氨苄西林耐药性最高;共130株菌株产生了多重耐药性（60.19%）,耐药菌株数最多的耐药谱为AMP-TET-STR,占比5.56%（12/216）,优势耐药谱不明显。结论 无锡市沙门菌的流行血清型以肠炎沙门菌和鼠伤寒沙门菌为主,且沙门菌耐药性形势严峻,应该尽快建立高效的管控机制, 加强药敏监测, 优化治疗方案, 避免抗生素滥用。     

Molecular characterization of antibiotic resistant Salmonella isolates from Indian foods

Salmonella isolates belonging to five serovars, Salmonella enterica Ohio, S. Oslo, S. Tennessee, S. Weltevreden and S. Typhimurium, isolated during 2006-2008 from food samples like sprouts and different varieties of fresh water and marine fish were tested for antibiotic resistance. High percentages (97%) of the isolates were resistant to at least one antibiotic and 82% of the isolates were resistant to more than one antibiotic. S. Oslo was the most resistant serovar and it exhibited resistance to 13 out of 16 antibiotics tested. Integron 1, which has been shown to confer multidrug resistance to various Salmonella serovars, was detected in multidrug resistant S. Oslo. PFGE studies revealed that serovars showed very high genetic diversity. The multidrug resistant S. Oslo showed unique PFGE pattern, which could be used in epidemiological studies.     

佳木斯市肠炎沙门菌的耐药特征及同源性分析

目的 了解佳木斯市优势血清型肠炎沙门菌的耐药状况及进化关系,为临床治疗提供科学依据。方法 对来自食品和腹泻患者的沙门菌进行血清学分型,采用微量肉汤稀释法对分离的肠炎沙门菌进行16种抗生素药敏试验,脉冲场凝胶电泳分析菌株之间的进化关系。结果 在108株沙门菌中,检出肠炎沙门菌55株（50.93%）;49株肠炎沙门菌有耐药性,耐药率为89.09%,其中33株菌（67.35%）为多重耐药,对抗生素萘啶酸和氨苄西林的耐药率较高,分别为81.82%和60.00%;55株肠炎沙门菌同源性为37%~100%。结论 佳木斯地区肠炎沙门菌多重耐药率高,菌株之间亲缘关系和耐药性存在密切关联性,来自食品和腹泻患者的肠炎沙门菌有交叉感染现象,依据本地区肠炎沙门菌耐药特征,制定有针对性诊疗方案和预防控制措施,对保障公共卫生健康发展具有重要意义。     

2016—2021年无锡市副溶血性弧菌毒力基因和耐药性分析及分子分型研究

目的 分析2016—2021年无锡市不同来源副溶血性弧菌的毒力基因、耐药性和分子分型结果。方法 采用多重荧光PCR、微量肉汤稀释法、脉冲场凝胶电泳（PFGE）分别对204株分离自无锡市各类监测样本中的副溶血性弧菌进行tlh/tdh/trh毒力基因检测、耐药试验和分子分型。数据比较采用χ²检验。结果 204株菌tlh基因携带率为100%（204/204）,tdh基因携带率为82.35%（168/204）,trh基因携带率为2.45%（5/204）,食品及环境分离株与病患分离株tdh基因携带率差异具有统计学意义（P<0.001）。菌株对头孢唑啉耐药率最高达96.08%（196/204）,对3种及以上抗菌药物的耐药率为2.94%（6/204）,食品及环境分离株与病患分离株对氨苄西林、四环素、磺胺甲??唑/甲氧苄啶、环丙沙星耐药率差异具统计学意义（P<0.05）;204株副溶血性弧菌经过聚类分析,分为123个PFGE带型,相似度49.1%~100.0%,按85%的相似度聚类可分为18个带型簇。结论 无锡市副溶血性弧菌病患分离株大部分携带tdh基因;菌株对头孢唑啉耐药率最高;PFGE型别呈多态性,优势带型不明显。