Sub-lethal damage of Listeria monocytogenes after long-term chilled storage at 4 degrees C

The possibility that long term in vitro chilled storage may result in sub-lethal damage to Listeria monocytogenes cells was investigated by comparing growth of chill-stored (starvation at 4 degrees C) and fresh cultures on selective and non-selective media. Growth of freshly grown cells was minimally (3-8%) affected by selective LSAMM agar compared with non-selective Brain Heart Infusion agar. In contrast, numbers of chill-stored strains were reduced by greater than 99% after direct plating on the same selective and non-selective media. Furthermore, chill-stored strains were able to grow in standard selective broth (Listeria Selective broth and Fraser broth) only if undiluted inocula (approximately 10(5)-10(6) cfu ml-1) were used, whereas they were capable of growth in Brain Heart Infusion broth even when the lowest dilutions were used (approximately 10(1) cfu ml-1). The potential public health consequences of this finding for the isolation of Listeria monocytogenes from foods is considered.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride.

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Exposure to salt and organic acids increases the ability of Listeria monocytogenes to invade Caco-2 cells but decreases its ability to survive gastric stress

The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37 degrees C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor sigmaB in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37 degrees C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).     

A Method for the Rapid Isolation of Listeria monocytogenes from Infected Material

Two media, one for enrichment and the other for differentiation of Listeria monocytogenes , are described and a method is proposed for the selective isolation of this bacterium from material containing a mixed bacterial flora such as faeces, vaginal swabs etc. Addition of potassium dichromate, chromium trioxide, thionin, nalidixic acid and amphotericin B to Todd-Hewitt Broth (BBL) made a satisfactory enrichment broth in which good selective growth of L. monocytogenes was obtained without notable damage to cells. The differentiation agar was Trypticase Soy Agar (BBL) supplemented with gallocyanin, pyronin and nalidixic acid. On this medium L. monocytogenes colonies, when viewed by the Henry's oblique transillumination technique, were blue in contrast to colonies of other bacterial species which were pink or red. Trials with experimentally infected materials showed that L. monocytogenes could be recovered from faeces infected with as few as 20 L. monocytogenes cells/g. All common contaminants, with the exception of a few strains of Streptococcus faecalis , were inhibited.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.     

Multiple deletions of the osmolyte transporters BetL,Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (DeltabetL DeltaopuC Deltagbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 micro mol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (DeltabetL Deltagbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 micro mol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.     

Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.

The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength. Previously, it was demonstrated that L. monocytogenes possesses an ATP-dependent L-carnitine transporter (A. Verheul, F. M. Rombouts, R. R. Beumer, and T. Abee, J. Bacteriol. 177:3205-3212, 1995). The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L. monocytogenes. The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium. Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute. Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine. The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane. Upon osmotic downshock, betaine and L-carnitine are rapidly released by L. monocytogenes as a consequence of activation of a channel-like activity. The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.     

Tolerance of acid-adapted and non-adapted Escherichia coli O157:H7 cells to reduced pH as affected by type of acidulant

A study was carried out to determine if three strains of Escherichia coli O157:H7 grown (18 h) in Tryptic Soy Broth (TSB) and TSB supplemented with 1.25% glucose (TSBG), i.e. unadapted and acid-adapted cells, respectively, exhibited changes in tolerance to reduced pH when plated on Tryptic Soy Agar (TSA) acidified (pH 3.9, 4.2, 4.5, 4.8, 5.1 and 5.4) with acetic, citric or malic acids. All test strains grew well on TSA acidified with acetic acid at pH > or = 5.4 or malic acid at pH > or = 4.5; two strains grew on TSA acidified with citric acid at pH > or = 4.5, while the third strain grew at pH > or = 4.8. Acid-adapted and control (unadapted) cells differed little in their ability to form visible colonies on TSA containing the same acid at the same pH. However, on plates not showing visible colonies, acid-adapted cells retained higher viability than unadapted cells when plated on acidified TSA. Growth of acid-adapted and control cells of E. coli O157:H7 inoculated into TSB containing acetic acid (pH 5.4 and 5.7) and citric or malic acids (pH 4.2 and 4.5) was also studied. There was essentially no difference in growth characteristics of the two types of cells in TSB acidified at the same pH with a given acid. Tolerance of acid-adapted and control cells on subsequent exposure to low pH is influenced by the type of acidulant. The order of sensitivity at a given pH is acetic > citric > malic acid. When performing acid challenge studies to determine survival and growth characteristics of E. coli O157:H7 in foods, consideration should be given to the type of acid to which cells have been exposed previously, the procedure used to achieve acidic environments and possible differences in response among strains. The use of strains less affected by pH than type of acidulant or vice versa could result in an underestimation of the potential for survival and growth of E. coli O157:H7 in acid foods.     

Growth of Listeria monocytogenes at different pH values in uncultured whey or whey cultured with Penicillium camemberti

Wheys from making Camembert cheese were either uncultured or cultured with Penicillium camemberti, adjusted to pH 5.0, 5.2, 5.4, 5.6, 6.2, and 6.8, and filter sterilized. Whey samples were inoculated to contain 100 to 500 Listeria monocytogenes (strains Scott A, V7, CA, or OH) cfu/mL and incubated at 6 degrees C. Counts of L. monocytogenes were obtained by surface plating appropriate dilutions on Tryptose Agar. Listeria monocytogenes failed to grow at or below pH 5.4; except for strains Scott A and OH which grew in cultured whey at pH 5.4 and attained populations of 7.8 x 10(3) and 5.4 x 10(4) cfu/mL, respectively, after 35 d of storage. In uncultured whey at pH 5.6, 6.2, and 6.8, populations of L. monocytogenes increased from 7.20 to 7.81, 7.51 to 8.23, and 7.48 to 8.08 log10 cfu/mL, respectively, after 35 d of storage at 6 degrees C. In cultured whey at pH 5.6, 6.2, and 6.8, numbers of L. monocytogenes increased from 7.53 to 8.13, 7.82 to 8.55, and 7.95 to 8.80 log10 cfu/mL, respectively, after 35 d of storage. Generation times for L. monocytogenes at 6 degrees C in uncultured whey at pH 5.6, 6.2, and 6.8 ranged between 25.3 and 31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respectively, depending on the Listeria strain. In contrast, generation times were significantly (p less than 0.05) shorter in cultured whey and ranged between 16.6 and 27.4 h, 10.3 and 16.6 h, and 17.4 and 16.3 h at pH values of 5.6, 6.2, and 6.8, respectively.     

The evaluation of amylolytic activity of strains of coagulase negative staphylococci

Amylase activity of 30 strains of Staphylococcus spp. was determined by Tryptic Soy Agar on supplemented with 1.0% starch as the substrate. After incubation (time incubation 24 h or 168 h), the plates were flooded with Lugol solution. A clear zone around the colonies indicated amylase activity. The 23 (76.7%) strains CNS demonstrated the amylase activity. It was observed that 17 (80.9%) strains of S. epidermidis, and 6 (66.7%) strains non-S. epidermidis, starch hydrolyzed. Amylase production depends of time incubation (frequently 168 h) and growth atmosphere (frequently oxygen atmosphere)     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Model systems allowing quantification of sensitivity to disinfectants and comparison of disinfectant susceptibility of persistent and presumed nonpersistent Listeria monocytogenes

Aims:  To determine if Listeria monocytogenes persistent strains differ from presumed nonpersistent strains in disinfection susceptibility and to examine the influence of attachment and NaCl on susceptibility.
Methods and Results:  Two model-systems that allowed quantitative inter-strain comparison of disinfectant sensitivity were developed. Persistent L. monocytogenes were not more tolerant to the disinfectants Incimaxx DES and Triquart SUPER than presumed nonpersistent isolates. When calibrating the systems with respect to presence of biological material and cell density, attached bacteria were as sensitive to disinfectants as were planktonic bacteria. Growth with 5% NaCl increased the tolerance of planktonic cells to Incimaxx DES. All strains of spot inoculated L. monocytogenes survived well 20 h of drying when protected by growth media and 5% NaCl, but were not protected by NaCl against disinfection.
Conclusions:  Persistent strains of L. monocytogenes are as susceptible to disinfectants as are presumed nonpersistent strains and attachment does not render the strains more tolerant to disinfectants. Growth with NaCl affected the susceptibility of the planktonic L. monocytogenes to Incimaxx DES and protected spot inoculated cells during drying.
Significance and Impact of the Study:  Attachment to surfaces does not per se offer protection to L. monocytogenes against disinfectants and disinfection tolerances do not appear to influence the ability of a strain to persist.     

Minimum water activity requirements for the growth of Listeria monocytogenes

The ability of five strains of Listeria monocytogenes to initiate growth at five different temperatures in brain heart infusion (BHI) broth adjusted to various water activity ( a _w) values with either sodium chloride (NaCl), sucrose or glycerol was investigated. Glycerol was the least toxic of the three solutes studied, with three of five strains of L. monocytogenes capable of growing in BHI broth adjusted with glycerol to an a w value of 0.90 at 30 C, compared to a w minima of 0.93 and 0.92 in broth adjusted with sucrose and sodium chloride, respectively. The minimum a w value required for growth generally increased as the incubation temperature decreased. Listeria monocytogenes appeared to tolerate glycerol and NaCl best when growing at 30 and 15°C, respectively, while for sucrose, temperature did not appear to influence growth of the organism. Listeria monocytogenes is one of the few food-borne pathogens that can grow at an a w value below 0.93.     

Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

VirAB在单核细胞增生李斯特菌耐药性及生物被膜形成中的作用

【背景】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)对一些临床常用抗生素、乳酸链球菌素(Nisin)等抗菌药物的敏感性下降,然而其背后的机制仍未完全阐明。【目的】调查转运蛋白VirAB在Lm对抗菌药物的耐药性及生物被膜形成中的作用。【方法】利用同源重组技术构建Lm基因缺失突变株,比较野生株和缺失株对抗菌药物的耐药性;利用微孔板法观测突变株生物被膜形成能力的变化;利用平板泳动法研究菌株的泳动能力。【结果】与野生株相比,virAB缺失突变株对头孢类抗生素、Nisin和溴化乙锭的敏感性增加;当培养基中分别添加亚致死浓度的苯扎氯铵、卡那霉素和四环素时,突变株均表现出不同程度的生长缺陷。缺失virAB后菌株形成生物被膜的能力下降。【结论】VirAB在Lm对头孢类等抗菌药物的耐药及生物被膜形成方面具有重要作用。     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.