Resistance mechanisms of bacteria to antimicrobial compounds

A range of antimicrobial compounds (bactericides) commonly termed biocides, microbicides, sanitizers, antiseptics and disinfectants are available, all of which are claimed by their producers to kill bacteria. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they are not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change, and where the biochemical basis is known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material. However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins, or to the development of resistance, either by adaptation or by genetic exchange. At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations. For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high bactericide concentration will kill bacteria with enhanced penetration barriers. It has been indicated that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations. Whereas the basis of bacterial resistance to antibiotics is well know, that of resistance to antiseptics, disinfectants and food preservatives is less well understood.Three mechanisms of resistance that have been reported include:

• limited diffusion of antimicrobial agents through the biofilm matrix,

• interaction of the antimicrobial agents with the biofilm matrix (cells and polymer),

• enzyme mediated resistance,

• level of metabolic activity within the biofilm

• genetic adaptation

• efflux pumps and

• outer membrane structure.

     

Kitchen hygiene in daily life

More and more data are available indicating that numerous (infectious) diseases are related to the home environment. Airborne microorganisms (bacteria, fungi), mites (in sheets or carpets), and (parts of) insects and beetles may be the cause of respiratory diseases such as asthma. In Europe and North America, more than half of the registered food infections appear to be contracted in the home. Then there is the development of new pathogens or the adaptation of microorganisms to extreme conditions. In many cases, the occurrence of adapted or resistant microorganisms is a reaction to the changing environment. Examples of this are the growth of Listeria monocytogenes in refrigerated food products and the presence of Legionella pneumophila in systems containing stagnant water.The main sources of infection in the domestic environment are people, pests, pets, and contaminated food and water. Germs are transmitted by direct contact with people or animals, by contaminated food, water, surfaces and air.Under circumstances favourable to microorganisms, the cells are able to survive or multiply into large numbers. Especially in places which stay moist for a long time considerable amounts of microorganisms are found, among with pathogenic types.The phrase ‘home hygiene’ does not merely refer to cleaning the house (daily). In practice, cleaning is not the only important issue; knowing how to prevent contamination is just as crucial. Domestic hygiene is the total sum of the measures used to prevent (insofar as possible) contamination with pathogens, and thus decreasing the number of infectious diseases. The hygiene measures required can be divided into three groups:

• Hygiene during food preparation.

• Personal and sanitary hygiene.

• Domestic environment.

This contribution will focus on food storage, food preparation and the effect of 'hygienic cleaners’ in the domestic kitchen.     

Polarity proteins regulate mammalian cell–cell junctions and cancer pathogenesis

Polarity pathways regulate important functions during the formation and maintenance of cell–cell junctions and during morphogenesis. In addition, cell polarity pathways are emerging as critical regulators of initiation and progression of carcinoma by functioning as tumor suppressors, downstream of oncogenes, or promoters of the metastatic process (Figure 2). It is highly likely that further analysis of cell polarity proteins and the pathways they control will identify novel biomarkers and potential drug targets for managing and treating patients with carcinoma.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

The role of β- and α-adrenoreceptors on blood flow and temperature of brown adipose tissue and involvement of nitric oxide in their effects

1. o

2. 1. Isoproterenol increased interscapular brown adipose tissue (BAT) blood flow and temperature.

3. 2. Phenylephrine increased BAT temperature, but did not affect blood flow. The effect was completely suppressed by N^ω-nitro- -arginine methyl ester ( -NAME), an inhibitor of endogenous NO synthesis.

4. 3. Isoproterenol plus phenylephrine increased BAT blood flow and temperature earlier than isoproterenol alone.

5. 4. CL316,243 increased BAT blood flow and temperature. These effects were also inhibited by -NAME.

6. 5. These data suggest that BAT blood flow as well as thermogenesis is regulated mainly by β-adrenoreceptors and partly by α₁-adrenoreceptors, and NO may be a common mediator for their regulations.

     

Metabolic rate, maximum metabolism, and advantages of torpor in the fat mouse Steatomys pratensis natalensis Roberts, 1921 (Dendeomurinae)

1. The fat mouse Steatomys pratensis natalensis (mean body mass 37.4±0.43 (se)) has a low euthermic body temperature T_b=30.1–33.8 °C and a low basal metabolic rate (BMR)=0.50 ml O₂ g⁻¹ h⁻¹.

2. Below an ambient temperature (T_a)=15 °C, the mice were hypothermic.

3. The lowest survivable T_a=10 °C.

4. Torpor is efficient in conserving energy between T_a=15–30 °C, below T_a=15 °C, the mice arouse.

5. Euthermic and torpid mice were hyperthermic at T_a=35 °C.

6. Thermal conductance was 0.159 ml O₂ g⁻¹ h⁻¹ °C⁻¹, 98.8% of the expected value.

7. Non-shivering thermogenesis (NST) was 2.196 ml O² g⁻¹ h⁻¹ (3.69×BMR).

8. Maximal oxygen consumption, however, was 3.83 ml O₂ g⁻¹ h⁻¹ (6.44×BMR), indicating that other methods of heat production are additive.

9. Because fat mice conserve energy by torpor only between T_a=15–30 °C, we suggest that torpor may be a more important mechanism for surviving food shortages than for surviving cold weather.

The effects of melanocortin agonists and antagonists on leptin-induced fever in rats

1. Over three experiments, separate groups of adult male Sprague–Dawley rats received intracerebroventricular (ICV) injections of either vehicle, recombinant rat leptin (1 μg), or leptin (4 μg), then two ICV injections, 30 min apart of vehicle/vehicle, leptin (4 μg)/vehicle, vehicle/α-MSH (300 ng), or leptin/α-MSH, and then vehicle/vehicle, leptin (4 μg)/vehicle, vehicle/ SHU-9119 (200 ng; a MC 3/4 receptor antagonist), or leptin/SHU-9119. Core temperatures (T_c), food intake and body weights were monitored.

2. Four microgram leptin resulted in the induction of fever, an effect blocked by injection of α-MSH. Antagonism of MC 3/4 receptors with SHU-9119 did not augment leptin-induced fever, but did block the inhibitory actions of leptin on food intake.

3. These data demonstrate the inhibitory effects of exogenous α-MSH on leptin-induced fever, but suggest that endogenous melanocortin action at MC 3/4 receptors does not tonically inhibit febrigenesis caused by leptin administration.

Low-temperature tolerance of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa

(1) The low-temperature tolerance of false codling moth (FCM) Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant aspect of this pest's population dynamics and has implications for post-harvest control and sterile insect technique programs.

(2) Here, we report results of experiments examining the effects of time, temperature and their interactions on low-temperature tolerance in adult FCM. In addition, using a variety of thermal pre-treatments, we examine the potential for hardening responses over several hours to improve low-temperature tolerance in FCM.

(3) Lower lethal temperature assays showed significant effects of time, temperature and significant interactions between time and temperature on survival (p<0.0001 in all cases). The temperature at which the probability of survival of 50% of the FCM population after 2 h of exposure was −4.5 °C, which varied significantly to −0.5 °C in 10 h. Gender and early adult age did not affect low-temperature tolerance of FCM.

(4) Using a range of non-lethal, low- and high-temperature pre-treatments, FCM survival could not be increased (p>0.84) and thus limited evidence for rapid cold hardening was found. These results are discussed with respect to microclimate temperatures in typical FCM environments and have implications for understanding population dynamics in this species and the diversity of low-temperature responses of insects.

Cooled neutrophils become deposited in the microcirculation after infusion: A potential mechanism for microvascular disruption following tissue hypothermia

1. We examined whether changes in rigidity and adhesiveness of neutrophils exposed to cooling and rewarming observed in vitro might impair microvascular perfusion in vivo. Neutrophils from donor rats were fluorescently (calcein-AM) or radioactively (Indium-111) labelled, incubated at 10 or 37 °C in vitro, and infused into recipients. Changes in transit rate and adhesive behaviour within post-capillary venules was quantified in m. extensor digitorum longus (EDL) using intravital microscopy, and tissue distribution determined.

2. There was an increased propensity of cooled cells to undergo adhesion following transfer into the recipient rat. However, cooling had no effect on median transit (354 μm s⁻¹) or rolling (14 μm s⁻¹) velocities during the first 5 min after infusion suggesting that cooling promotes adhesion, but does not delay passage through capillaries. Cooled neutrophils subsequently transformed to stationary adhesion. Their immobilisation was higher than for cells held at 37 °C (P<0.05), and once immobilised they remained firmly adherent to the vessel wall. Cooled, radiolabelled neutrophils showed tissue-specific accumulation after 3 min, but were cleared to the same extent as warmed cells by 20 min.

3. Our data suggest that cooling and rewarming of neutrophils impairs their ability to transit microvessels, reflecting changes in adhesive and mechanical properties observed in vitro, and may contribute to cold-associated circulatory pathology.

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M_w and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively.The solution conformation of chitosan was then estimated from:

(a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [η] = kM_w^a and

(b) the persistence length, L_p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475].

Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (L_p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.     

Assessment of the efficacy of disinfectants on surfaces

The Literature on testing the efficacy of disinfectants covers a century. Most predominant and standardized are the so called suspension tests that allow for the quantitative estimation of the microbicidal activity (log reduction factors) of disinfectants on test organisms suspended in solutions of these products.Since the outcome of suspension tests might be a poor predictor for the efficacy of a disinfectant under practical circumstances, especially with regard to bacteria attached to surfaces, a variety of test procedures have been designed to mimic those conditions. Within the framework of CEN/TC 216 a quantitative surface test has been developed to assess the activity of disinfectants on bacteria or fungi attached to steenless steel surfaces. Preliminary data suggest that covering a dried inoculum with disinfectant without any further mechanical action to improve contact between organisms and disinfectant, will usually result in lower reduction factors than those obtained with suspension tests. Comparative testing further suggests that by applying mechanical action, with the effect of resuspending cells in the liquid on the surface,—similar to mopping, brushing etc.— will result in higher reduction rates. Although not unexpected these findings emphasize the importance of designing test methods based on practical applications of disinfectants.     

Benthic foraminiferal associations in the Northern Bay of Safaga, Red Sea, Egypt

In the shallow-water area (0–70 m) of the Northern Bay of Safaga, 73 surface samples were studied with respect to total foraminiferal fauna. The samples cover a great variety of shallow-water environments and yielded 239 foraminiferal taxa. Based on q-mode cluster analysis, and tested by canonical discriminant analysis, the samples are grouped into 13 foraminiferal associations, each characterized by several species:

(1) Quinqueloculina mosharrafai-Borelis schlumbergeri-Brizalina simpsoni Ass.,

(2) Heterostegina depressa-Amphlstegina lessonii/bicirculata Ass.,

(3) Cibicidids-Rosalina-Amphistegina lobifera-Pseudoschlumbergerina ovata Ass.,

(4) Peneroplis planatus Ass.,

(5) Peneroplis planatus-Varidentella neostriatula Ass.,

(6) Peneroplis planatus-Coscinospira hemprichii-Varidentella neostriatula Ass.,

(7) Quinqueloculina spp. Ass.,

(8) Hauerina diversa-Sorites orbiculus Ass.,

(9) Verneuilina sp.-n Articulina pacifica-Reussella simplex Ass.,

(10) Textularia agglutinans/rugulosa-Bolivina variabilis Ass.,

(11) Textularia agglutinans-Challengerella bradyi-Elphidium jenseni/simplex Ass.,

(12) Operculina ammonoides-Adelosina laevigata-Brizalina striatula/subspathulata Ass.,

(13) Bolivina variabilis—Miliolinella-Nonion fabum-Elphidium simplex Ass.

The characteristic forms of each association are linked to specific environmental parameters and modes of life. As long as taxonomic uniformitarianism can be applied, similar associations with comparable ecological demands may also be detected in the fossil record. For comparisons with taxonomically different material the 13 associations are summarized into 4 categories each reflecting a specific type of substrate. In the fossil record these categories may be recognized by general morphological characteristics of the foraminiferal tests and by sedimentological data. The 4 substrate types are: hardground, sand (with or without seagrass and/or corals), firmground, and soft bottom.     

Acute effect of a single whole-body cryostimulation on prooxidant–antioxidant balance in blood of healthy, young men

1. We have examined the prooxidative–antioxidative reaction to extremely low temperatures (−130 °C) during a one-time cryostimulation in 15 young, clinically healthy individuals.

2. The total lipid peroxides as the total oxidative status (TOS) and the total antioxidative status (TAS) were measured in blood plasma collected in the morning of the day of cryostimulation, 30 min after the cryostimulation, and on the following morning.

3. The level of stress expressed by total oxidative status in plasma, resulting from exposure to extremely low temperatures, was statistically significantly lowered 30 min after leaving the cryochamber than prior to the exposure. The next day, the TOS level still remained lower than the initial values. The TAS level decreased after leaving the cryochamber and remained elevated the following day.

Bioenergetic responses of sub-adult sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea) to temperature with special discussion regarding its southernmost distribution limit in China

1. This paper investigated the bioenergetic responses of the sea cucumber Apostichopus japonicus (wet weights of 36.5±1.2 g) to different water temperatures (5, 10, 15, 20, 25 and 30 °C) in the laboratory.

2. Results showed that theoretically the optimal temperatures for energy intake and scope for growth (SFG) of sub-adult A. japonicus was at 15.6 and 16.0 °C, respectively. The aestivation threshold temperature for this life-stage sea cucumber could be 29.0 °C by taking feeding cessation as the indication of aestivation.

3. Our data suggests that A. japonicus is thermo-sensitive to higher temperature, which prevents it from colonising sub-tropical coastal zones. Therefore, water temperature plays an important role in its southernmost distribution limit in China.

4. The potential impact of global ocean warming on A. japonicus might be a northward shift in the geographical distribution.

Antioxidative defense organization in retroperitoneal white adipose tissue during acclimation to cold—The involvement of l-arginine/NO pathway

1. Retroperitoneal white adipose tissue (RpWAT) antioxidative defense was investigated in untreated, l-arginine-treated and N^ω-nitro-l-arginine methyl ester (l-NAME)-treated rats kept at 4±1 °C (1, 3, 7, 12, 21 and 45 days) and compared to control rats at 22±1 °C.

2. Cold-acclimation-induced RpWAT weight decrease was accompanied by a decline in glutathione level and increased activity of manganese superoxide dismutase (MnSOD), glutathione S-transferase (GST), catalase, glutathione peroxidase and glutathione reductase at different time-points.

3. l-arginine accelerated RpWAT weight decrease, the increase in MnSOD and GST activities and the prolonged increase of catalase, MnSOD and GST activities. l-NAME delayed cold-induced catalase activity increase and tissue weight decrease. Prolonged l-NAME-treatment had a similar effect on RpWAT as l-arginine.

4. Results suggest the involvement of l-arginine/NO pathway in RpWAT oxidative metabolic augmentation induced by cold-acclimation.

Pulmonary gas exchange kinetics during exercise: physiological inferences of model order and parameters

1. 1. The ventilatory and pulmonary gas exchange responses during moderate exercise can be appropriately modelled with first-order dynamics.

2. 2. A delay term, reflecting tissue-to-lung transit time, is needed for accurate characterization, however.

3. 3. The O₂ uptake time constant ( reflects the enzymatically controlled tissue O₂ utilization.

4. 4. is appreciably longer than , consequent to the tissue CO₂ capacitance.

5. 5. As typically longer than , transient errors in alveolar and arterial blood gas tensions are predicted: small for P_CO2 but much larger for P_O2.

6. 6. At work rates above the lactate threshold, a slow and delayed component of V̇_O2 induces an additional V̇ component (“excess” V̇_O2), leading to more rapid fatigue.

7. 7. The ventilatory compensation for the metabolic acidemia at these work rates is slow, with compensation being poor for rapid-incremental exercise.

8. 8. A justifiable control model of the coupling of ventilation to metabolism must cohere with these demonstrable physiological characteristics.

Alternative production process strategies in E. coli improving protein quality and downstream yields

Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g⁻¹ DCW, but the final specific enzyme activity (1.7 AU mg⁻¹ RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield:

(i) Temperature reduction to 28 °C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g⁻¹ DCW. The enzyme activity increased to 4 AU mg⁻¹ RhuA and purification-immobilization yield to 93%.

(ii) A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g⁻¹ DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg⁻¹ RhuA, and 95% of immobilization yield was achieved.

For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved.     

Determination of ochratoxin A in liquorice products using HPLC-based analytical methods. Part II: harmonised method and method validation study

On the basis of the outcome of an European proficiency test series conducted on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A, a new harmonised method was developed for the analysis of ochratoxin A in liquorice extracts. This method works without the use of halogenated solvents because, as the proficiency test showed, an aqueous extraction solution can be used instead of, for example, chloroform, whose use is restricted in the EU. The main objective of this method validation study was to check the performance of this harmonised method. To carry out the method validation study, a set of three different test samples (one liquorice powder and two liquorice pastes) and a liquorice powder sample with an indicated range of ochratoxin A (a so-called sunshine sample) was distributed to 21 laboratories in ten countries throughout Europe and to one laboratory in the USA. The study was evaluated according to internationally recognised guidelines. In terms of its repeatability and reproducibility for determining ochratoxin A in liquorice extracts with a relative standard deviation for repeatability (RSD_r) of between 6.68 and 19.95 and a relative standard deviation for reproducibility (RSD_R) of between 17.39 and 29.08 the performance of the harmonised method was found to be in the accepted range of the EU directive for the analysis of mycotoxins in several foodstuffs.