The effect of physical and microbiological factors on food container leakage

The effect of physical and microbiological factors on food container leakage was investigated in a container leakage model system (CLMS). The leakage of Acineto-bacter calcoaceticus, Staphylococcus sp., Pseudomonas sp., Bacillus sp., a coryneform, Staph. aureus , and two biotest organisms ( Enterobacter cloacae NC1B 8151 and Ent. aerogenes MB31) was studied. The rate of bacterial leakage (log₁₀ cells/channel/s) was greater in the presence of a partial vacuum of 51–305 mm Hg than at atmospheric pressure. Fluid flow (ml) through leakage channels was increased by the application of vacuum. Leakage varied with vacuum, bacterial morphology, cell concentration, leakage channel size (0.78–120 μm²) and channel shape (straight or convoluted). The number of leaked cells was not proportional to vacuum or channel size. The effect of channel shape varied with bacterial species. Increased container medium viscosity decreased bacterial leakage. Fluid flow through leakage channels was generally reduced by the most viscous solution. Cells from biofilms and mono-layers of Ac. calcoaceticus or Staph. aureus attached to nylon (Hyfax) or stainless steel surfaces underwent leakage. Mixed bacterial populations had characteristic leakage rates against vacuum different from the leakage pattern of individual species in the population. The composition of the leaked population was different from the original inoculum. The results indicated that container leakage is a complex process involving a range of interdependent factors.     

The effect of temperature and relative humidity on the survival of bacteria attached to dry solid surfaces

The effect of relative humidity (r.h.) (0%, 34% and 75%) and temperature (4, 15 and 25°C) on the desiccation survival of Pseudomonas sp., Acinetobacter calcoaceticus , a coryneform, Staphylococcus sp., and Staphylococcus aureus attached to and grown on glass surfaces was studied. The survival of Gram-negative and Gram-positive species was similar. Bacterial survival was shortest at 25°C for all species studied, and in most cases longest at 4°C. The effect of r.h. on survival varied with species. The number of microcolonies and the number of cells within microcolonies for Pseudomonas sp. increased on the dry surfaces after 7 d incubation under all desiccation conditions.     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

THE INFLUENCE OF CERTAIN MANUFACTURING PROCESSES ON THE STAPHYLOCOCCUS AUREUS CONTENT OF SPRAY-DRIED MILK

SUMMARY: Dried milk was prepared from milks inoculated with pure cultures of Staph. aureus , using a small pilot-type batch vacuum evaporator and spray drier. Vacuum evaporation reduced the numbers of Staph. aureus in most experiments but one strain was capable of multiplication at evaporating temperatures up to about 43°. Above 49° the organisms declined rapidly and at 56° they were eliminated. Residual staphylococci present in the warm concentrated milk feeding the drier increased in some experiments but declined markedly in others. Staphylococci added directly to warm concentrated milk always proliferated. A small proportion of staphylococci survived spray drying, even with high hot air temperatures. Different strains varied in their susceptibility to different stages of the whole process.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Cellular effects of monohydrochloride of L-arginine, N-lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus

AIMS: Here we study the effect of monohydrochloride of L-arginine, N(alpha)-lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 microg ml(-1), respectively. METHODS AND RESULTS: Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane. Cell integrity was altered mainly in the outer membrane of S. typhimurium, but there was no significant change in the cytoplasm. However, in Staph. aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm. Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis. In S. typhimurium the proportion of damaged cells after 24 h was 97% and in Staph. aureus 56.3%. LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7.7 and 3.34 microg ml(-1) for Staph. aureus and S. typhimurium, respectively. Membrane disruption was detected by measuring the proton flow across the membrane. CONCLUSIONS: Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time the cellular effects of LAE on bacterial cells were studied.     

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.     

Biofilm interactions between distinct bacterial genera isolated from drinking water

In the environment, multiple microorganisms coexist as communities, competing for resources and often associated as biofilms. In this study, single- and dual-species biofilm formation by, and specific activities of, six heterotrophic intergeneric bacteria were determined using 96-well polystyrene plates over a 72-h period. These bacteria were isolated from drinking water and identified by partial 16S rRNA gene sequencing. A series of planktonic studies was also performed, assessing the bacterial growth rate, motility, and production of quorum-sensing inhibitors (QSI). This constituted an attempt to identify key attributes allowing bacteria to effectively interact and coexist in a drinking-water environment. We observed that in both pure and dual cultures, all of the isolates formed stable biofilms within 72 h, with specific metabolic activity decreasing, in most cases, with an increase in biofilm mass. The largest single- and dual-biofilm amounts were found for Methylobacterium sp. and the combination of Methylobacterium sp. and Mycobacterium mucogenicum, respectively. Evidences of microbial interactions in dual-biofilm formation, associated with appreciable biomass variation in comparison with single biofilms, were found for the following cases: synergy/cooperation between Sphingomonas capsulata and Burkholderia cepacia, S. capsulata and Staphylococcus sp., and B. cepacia and Acinetobacter calcoaceticus and antagonism between S. capsulata and M. mucogenicum, S. capsulata and A. calcoaceticus, and M. mucogenicum and Staphylococcus sp. A neutral interaction was found for Methylobacterium sp.-M. mucogenicum, S. capsulata-Staphylococcus sp., M. mucogenicum-A. calcoaceticus, and Methylobacterium sp.-A. calcoaceticus biofilms, since the resultant dual biofilms had a mass and specific metabolic activity similar to the average for each single biofilm. B. cepacia had the highest growth rate and motility and produced QSI. Other bacteria producing QSI were Methylobacterium sp., S. capsulata, and Staphylococcus sp. However, only for S. capsulata-M. mucogenicum, S. capsulata-A. calcoaceticus, and M. mucogenicum-Staphylococcus sp., dual-biofilm formation seems to be regulated by the QSI produced by S. capsulata and Staphylococcus sp. and by the increased growth rate of S. capsulata. The parameters assessed by planktonic studies did not allow prediction and generalization of the exact mechanism regulating dual-species biofilm formation between the drinking-water bacteria.     

The antimicrobial activity of Fleroxacin RO 23-6240, a third generation fluoroquinolone derivative, tested in vitro

The broad antimicrobial spectrum of Fleroxacin RO 23-6240 was tested on a panel of bacterial and several mycobacterial species. Staph. aureus strains, gramnegative bacteria E. coli, Kl. pneumoniae, Salmonella spp. were found to be rather susceptible to the tested preparation in vitro. The same was true of those species which are known to feature strong natural resistance, e.g. Ps. aeruginosa and Acinetobacter calcoaceticus. As regards mycobacteria, complete susceptibility was recorded for 10 M. tuberculosis strains, 17 M. kansasii strains and 4 strains of M. fortuitum. Only two of the eleven tested strains of the complex M. avium-intracellulare were found to be susceptible and one M. chelonae strain tested proved resistant. Due to their broad spectrum and strong bactericidal effects, chinolone preparations can be anticipated to assume an everincreasing significance in the future.     

The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.     

Growth and survival of bacteria implicated in sudden infant death syndrome on cot mattress materials

AIMS: To compare growth and survival of selected bacteria implicated in sudden infant death syndrome (SIDS) on cot mattress polyurethane (PU) inner-foams and on different types of cot mattress cover materials. METHODS AND RESULTS: Escherichia coli, Staphylococcus aureus or Streptococcus pyogenes were inoculated onto swatches of new-unused cot mattress PU inner-foam and onto three types of cot mattress covers (polyvinyl chloride, cotton and polyester). The influence of inoculation cell density, relative humidity (RH) and temperature of incubation on survival was assessed by recovery of cells in 0.85% NaCl, with viable cell enumeration by plate counting on selective and differential media. Utilization of carbon and nitrogen sources within cot mattress PU was assessed by following growth on aqueous leachate from PU, and by colorimetric determination of aromatic amines. Good survival capability (>206 d) was shown by all three test species on PU inner-foam and on polyester mattress cover at high RH (75%), but only by Staph. aureus on PU at low RH (25%). Aqueous soluble material from PU foam supports bacterial growth; removal of aromatic amines from aqueous leachate from PU accompanies growth of Staph. aureus. CONCLUSIONS: Staphylococcus aureus has good survival capability on cot mattress PU foam, even at low RH. Soluble material within PU can serve as carbon and nitrogen sources for bacterial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Prolonged survival of Staph. aureus on PU at low RH could explain, in the context of the common bacterial toxins hypothesis, an increased risk of SIDS associated with used infant mattresses.     

Effect of detergents on the chloramphenicol inactivation process by resistent bacteria

The effect of detergents, i. e. cationic, anionic, nonionic and polyelectrolytes of the cationic type on the efficacy of chloramphenicol against resistant strains of E. coli and Staph. aureus was studied. It was found that the detergent effect on inactivation of chloramphenicol by the bacterial resistant strains was inconsistent. The cationic detergents and in particular chlorhexidine had the most pronounced inhibitory effect. In subbacteriostatic concentrations they significantly suppressed inactivation of chloramphenicol in the cells of E. coli and Staph. aureus. The anionic detergents and polyelectrolytes of the cationic type in the above concentrations were effective only with respect to Staph. aureus. It is noted that the detergents increased the activity of chloramphenicol against E. coli and Staph. aureus.     

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil

Aims:  Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213.
Methods and Results:  The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa , membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus .
Conclusions:  Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed.
Significance and Impact of the Study:  Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.     

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

Damage to Escherichia coli on Exposure to Moist Heat

The effects of temperatures of 50 to 60 C on suspensions of a strain of Escherichia coli are described. At these temperatures, the substances which leaked from the cells were determined as pentoses; the amount leaked over a 30-min period increased with increasing temperature. The leakage materials from suspensions heated in water, sodium chloride, sucrose, and sucrose plus Mg(++) were examined spectrophotometrically, and the ratios of the absorbances at 280 and 260 nm (the 280 to 260 nm ratio) were determined. It was not possible to determine protein by this method, as the ratio was always <0.7. When saline was the suspending medium, the ratio was lower than when water was used, and there was a greater leakage of 260 nm absorbing material. When suspended in sucrose or in sucrose plus Mg(++), penicillin-induced spheroplasts did not undergo lysis, but became less regular in shape, and there was an increase in the extinction at 500 nm. The overall effects of high temperatures on nonsporeforming bacterial cells are discussed; in view of the available evidence, it is concluded that ribonucleic acid degradation is an event which is probably closely related to thermally induced bacterial death.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

"Methicillin-resistant" Staphylococcus aureus: reassessment by controlled trial in burns unit.

A controlled trial of oral flucloxacillin (250 mg six-hourly for four days) was performed in 34 patients treated by the covered method whose burns had yielded a heavy or moderate growth of Staphylococcus aureus resistant to methicillin at 30 degrees C but moderately sensitive at 37 degrees C. Staph aureus was eliminated in nine of the 17 patients treated with flucloxacillin but in none of the 17 controls; the proportion of patients from whose burns sensitive Staph aureus was eliminated in an earlier trial of cloxacillin was greater than this. Strains of Staph aureus commonly described as methicillin-resistant and showing heterogeneous growth at 37 degrees C of many sensitive and very few resistant bacterial cells should, in the light of these findings, be called moderately sensitive to flucloxacillin. Such "heteroresistant" strains showed consistent moderate sensitivity in replicate diffusion sensitivity tests at 37 degrees C, but very inconsistent results in replicate dilution tests, especially with flucloxacillin. These studies showed that 18-hour diffusion sensitivity tests indicate the clinical value of treatment with flucloxacillin for staphylococcal infections of moderate severity more correctly at 37 degrees C than at 30 degrees C.     

Clinical prevalence and antimicrobial susceptibility of Staphylococcus aureus and Staph. intermedius in dogs

AIMS: This study was undertaken to investigate whether the antibiotic resistance of Staphylococcus aureus and Staph. intermedius varies with the site of isolation, sex or age of dogs. METHODS AND RESULTS: A total of 867 isolates of Staph. aureus and 1339 isolates of Staph. intermedius were obtained from nose, eye, ear, reproductive extremity, urine, abscess, skin and throat isolates. Staphylococcus intermedius isolates were isolated most frequently and adult and male dogs were more common compared with juveniles and/or female dogs. Antimicrobial resistance was commonly found for penicillin G, lincomycin, tetracycline and trimethoprim-sulphamethoxazole in both Staphylococcus species. Surprisingly, we detected significant resistance to cloxacillin in male (67.1%) and female (69.4%) Staph. aureus isolates, irrespective of the anatomical site of isolation. The resistance or susceptibility of isolates of Staph. aureus from reproductive extremities and isolates of Staph. intermedius from ear, eye and abscess sites was associated with the age of the animal. CONCLUSIONS: Antimicrobial susceptibilities in Staph. aureus and Staph. intermedius often differed with regard to the site of isolation, sex and age of the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing antimicrobial resistance in staphylococci in veterinary medicine complicates the empirical selection of antimicrobial agents. These complications reveal a continuously evolving, complicated multifactoral process of the site of isolation, sex and age of the animal.