Apostolos S. Angelidis

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 stx(2)-positive, five of which were also stx(1)-positive. The remaining isolate was positive only for the stx(1) gene. All stx-positive isolates (whether positive for stx(1), stx(2) or stx(1) and stx(2)) 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.

Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization (FISH)

The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organisms fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.

Loss of viability of Listeria monocytogenes in contaminated processed cheese during storage at 4, 12 and 22 °C

The behaviour of Listeria monocytogenes in a processed cheese product was evaluated over time by inoculating the product with three different L. monocytogenes strains (Scott A, CA and a strain isolated from processed cheese) at three different inoculation levels (ca. 6x10(5), ca. 6x10(3) and 10(2)CFU/g of cheese or less) and after storage of the contaminated products at 4, 12 or 22 degrees C. Growth of L. monocytogenes was not observed in any of the experimental trials (experiments involving different combinations of strain, inoculum level and storage temperature) throughout the storage period. L. monocytogenes populations decreased over time with a rate that was strain- and storage temperature-dependent. Nonetheless, for cheeses that had been inoculated with the higher inoculum and stored at 4 degrees C viable populations of L. monocytogenes could be detected for up to nine months post-inoculation. The L. monocytogenes survival curves obtained from the different trials were characterised by a post-inoculation phase during which the populations remained essentially unchanged (lag phase) followed by a phase of logarithmic decline. The duration of the lag phase and the rate of inactivation of L. monocytogenes in the different trials were estimated based on data from the linear descending portions of the survival curves. In addition, a non-linear Weibull-type equation was fitted to the data from each survival curve with satisfactory results. The results of the present study emphasize that, according to the definition laid down in the European Union Regulation 1441/2007, the processed cheese product tested in this work should be considered and classified as one that does not support the growth of L. monocytogenes under reasonable foreseeable conditions of distribution and storage. However, post-processing contamination of the product should be austerely avoided as the pathogen can survive in the product for extended periods of time, particularly under refrigerated storage (4 degrees C).

Methicillin-resistant Staphylococcus aureus: a controversial food-borne pathogen

Methicillin‐resistant Staphylococcus aureus (MRSA) is a major cause of severe healthcare‐associated (HA) infections. Although during the last decade the incidence of HA invasive infections has dropped, the incidence of community‐associated MRSA (CA‐MRSA) infections has risen among the general population. Moreover, CA‐MRSA, livestock‐associated MRSA (LA‐MRSA) and HA‐MRSA (HA‐MRSA) can be found in foods intended for human consumption. Several studies from different geographical areas have reported the presence of enterotoxin genes in several MRSA food isolates. Molecular typing studies have revealed genetic relatedness of these enterotoxigenic isolates with isolates incriminated in human infections. The contamination sources for foods, especially animal‐origin foods, may be livestock as well as humans involved in animal husbandry and food‐processing. Under favourable environmental conditions for growth and enterotoxin production, enterotoxigenic S. aureus isolates present in foods can cause staphylococcal food poisoning (SFP), irrespective of the contamination origin. Owing to the typically moderate clinical manifestations of SFP, the S. aureus strains responsible for SFP (cases or outbreaks) are frequently either not identified or not further characterized. Antimicrobial susceptibility testing is rarely performed, because administration of antimicrobial therapy is not required in the vast majority of cases. Staphylococcal food poisoning is the result of consumption of foods with preformed enterotoxins. Hence, similar to methicillin‐sensitive enterotoxigenic S. aureus, enterotoxigenic MRSA can also act as food‐borne pathogens upon favourable conditions for growth and enterotoxin production. The severity of the intoxication is not related to the antimicrobial resistance profile of the causative S. aureus strain and therefore MRSA food‐borne outbreaks are not expected to be more severe.

Bacterial subclinical mastitis and its effect on milk yield in low-input dairy goat herds.

The objectives of this study were (1) to record the major pathogens associated with subclinical mastitis (SCM), (2) to calculate their incidence during the milking period, and (3) to estimate the effect of SCM on daily milk yield (DMY) for goats reared under low-input management schemes. Dairy goats (n=590) of Skopelos and indigenous Greek breeds from 4 herds were randomly selected for the study. The study included monthly monitoring, milk yield recording, and bacteriological analyses of milk of individual goats during the course of 2 successive milking periods. Incidence and cumulative incidence were calculated for SCM cases. Moreover, 2 mixed linear regression models were built to assess the effects of (1) SCM and (2) different pathogens isolated from SCM cases, on DMY. The estimated incidence and cumulative incidence of SCM for the first and the second year of the study were 69.5 and 96.4 new cases of SCM/1,000 goat-months, and 24.1 and 31.7%, respectively. A total of 755 milk samples were subjected to microbiological examination, resulting in 661 positive cultures. Coagulase-negative and coagulase-positive staphylococci were isolated from 50.2 and 34.5% of the positive cultures, respectively. The incidence of infections (new infections per 1,000 goat-months) for the first and the second year of the study were 34 and 53 for coagulase-negative staphylococci, 23 and 28 for coagulase-positive staphylococci, 3 and 5 for Streptococcus/Enterococcus spp., and 5.5 and 9.1 for gram-negative bacteria. Goats with SCM had lower DMY when compared with goats without SCM (ca. 47g/d, corresponding to a 5.7% decrease in DMY). In particular, goats with SCM due to coagulase-positive staphylococci infection produced approximately 80g/d less milk (a reduction of ca. 9.7%) compared with uninfected ones, whereas SCM due to gram-negative bacteria resulted in approximately 15% reduction in DMY. Investigating the epidemiology of SCM and its effects on production traits is critical for the establishment of effective preventive measures against SCM and for the assessment of the sustainability of production in low-input dairy goat herds.

Growth of Aeromonas hydrophila in the Whey Cheeses Myzithra, Anthotyros, and Manouri during Storage at 4 and 12°C

The fresh whey cheeses Myzithra, Anthotyros, and Manouri were inoculated with Aeromonas hydrophila strain NTCC 8049 (type strain) or with an A. hydrophila strain isolated from food (food isolate) at levels of 3.0 to 5.0 × 102 CFU/g of cheese and stored at 4 or 12°C. Duplicate samples of cheeses were tested for levels of A. hydrophila and pH after up to 29 days of storage. At 4°C, A. hydrophila grew in Myzithra and Anthotyros with a generation time of ca. 19 h, but no growth was observed in Manouri. In Myzithra, average maximum populations of 8.87 log CFU/g (type strain) and 8.79 log CFU/g (food isolate) were recorded after 20 and 22 days of storage at 4°C, respectively. The average maximum populations observed in Anthotyros stored at 4°C were 6.72 log CFU/g (food isolate) and 6.13 log CFU/g (type strain) and were observed after 15 and 16 days of storage, respectively. A. hydrophila grew rapidly and reached high numbers in cheeses stored at 12°C. The average generation times were 3.7 and 3.9 h (Myzithra), ...

Bi-phasic growth of Listeria monocytogenes in chemically defined medium at low temperatures.

The present work reports a novel observation regarding the growth of L. monocytogenes in modified Welshimers broth (MWB) at low temperatures. Specifically, the direct monitoring of the growth of L. monocytogenes Scott A using plate count data revealed that the pathogen displays a bi-phasic growth pattern in MWB at 7 °C. This bi-phasic growth pattern is masked (not observed) when optical density (OD) measurements are used to monitor growth due to the inability of OD readings to detect L. monocytogenes population density increases up to 10(7) CFU/mL. This bi-phasic growth phenomenon was further investigated as a function of growth temperature (4 °C, 7 °C, 10 °C, 14 °C and 18 °C), medium composition (by altering the MWB composition by ten-fold increases in different sets of medium constituents), inoculum level (10(2), 10(3), 10(4), 10(5), 10(6), and 10(7) CFU/mL) and L. monocytogenes strain (10 strains). The growth of L. monocytogenes Scott A in MWB at 7 °C, 10 °C and 14 °C was consistently bi-phasic and independent of growth rate; at 18 °C, growth was consistently mono-phasic (single-phase, typical sigmoid growth curves), whereas no growth was observed at 4 °C. The tested modifications in the composition of MWB did not influence the bi-phasic nature of L. monocytogenes Scott A growth at 7 °C, and, overall, we could not point out any strain-, or serotype-specific effects. On the other hand, the initial inoculum level appears to affect the form of the growth curve, as there was a shift towards mono-phasic growth in trials with increasing initial inocula. A mathematical model, based on a stepwise response and described through two sequential sigmoid curves, was used to describe bi-phasic growth and estimate the kinetic parameters of L. monocytogenes growth. An alternative hypothesis, based on the assumption of the existence of two subpopulations, possessing different growth kinetics, materialized under the stress imposed on L. monocytogenes cells due to the combined effect of three factors (defined medium, low temperature and low initial inoculum) was also proposed and formulated.

The microbiological quality of pasteurized milk sold by automatic vending machines.

The microbiological quality of pasteurized milk samples (n = 39) collected during 13 weekly intervals from three automatic vending machines (AVM) in Greece was investigated. Microbiological counts (total aerobic (TAC), total psychrotrophic (TPC), Enterobacteriaceae (EC), and psychrotrophic aerobic bacterial spore counts (PABSC)) were obtained at the time of sampling and at the end of shelf‐life (3 days) after storage of the samples at 4 or 8°C. TAC were found to be below the 107 CFU ml−1 limit of pasteurized milk spoilage both during sampling as well as when milk samples were stored at either storage temperature for 3 days. Enterobacteriaceae populations were below 1 CFU ml−1 in 69·2% of the samples tested at the time of sampling, whereas the remaining samples contained low numbers, typically less than 10 CFU ml−1. All samples tested negative for the presence of Listeria monocytogenes. Analogous microbiological data were also obtained by sampling and testing prepackaged, retail samples of pasteurized milk from two dairy companies in Greece (n = 26). From a microbiological standpoint, the data indicate that the AVM milk samples meet the quality standards of pasteurized milk. However, the prepackaged, retail milk samples yielded better results in terms of TAC, TPC and EC, compared to the AVM samples at the end of shelf‐life.

Bacterial subclinical mastitis and its effect on milk quality traits in low-input dairy goat herds

The objective of the study was to investigate and quantify the effects of subclinical mastitis (SCM) on the gross chemical composition of milk in low-input dairy goat herds. Dairy goats (n=590) of two native Greek breeds from four representative low-input farms were randomly selected and used in the study. Α prospective study was conducted, including monthly monitoring and milk sampling of the same individual goats during the course of two consecutive milking periods. Mixed linear regression models were built to assess how the chemical composition of milk was affected by (1) SCM and (2) the different pathogens isolated from SCM cases. Goats with SCM had lower milk-fat content (MFC), daily milk-fat yield (DMFY), milk-lactose content (MLC) and daily milk-lactose yield (DMLY), and slightly higher milk-protein content (MPC) and daily milk-protein yield (DMPY), compared with goats without SCM. Milk produced by goats with SCM due to coagulase-positive staphylococci and Mycoplasma agalactiae had significantly lower MFC, DMFY, MLC and DMLY, and higher MPC and DMPY, compared with the milk produced by healthy goats. Finally, goats with SCM due to coagulase-negative staphylococci had lower DMFY, MLC and DMLY and higher DMPY compared with the healthy ones.

Applicability of the EN ISO 11290-1 standard method for Listeria monocytogenes detection in presence of new Listeria species

During the past six years, new species of the genus Listeria have been isolated from foods and other environmental niches worldwide. The Standard method EN ISO 11290-1 that is currently under revision will include in its scope all Listeria species in addition to L. monocytogenes. The objective of this project was to evaluate the ability of the Standard EN ISO 11290-1 method to detect and identify the newly discovered Listeria spp., and to assess potential over-growth effects of the new species in mixed cultures with L. monocytogenes during each step of the enrichment process. This objective was addressed by the generation of necessary data on the behavior of the new species during the pre-enrichment and the enrichment steps of the reference method as well as data on their phenotypic characteristics on rich and selective media used for isolation and identification. Most of the new Listeria species developed well on selective agar media for Listeria, however the recovery of some species was difficult due to poor growth in Half Fraser and Fraser broth. Good results (consistently positive) were obtained for confirmation at the genus level via the catalase test, the Gram test and the blueish appearance test on non-selective medium, but not with the VP test, as most of the new species yielded a negative result. In the light of results obtained in co-culture experiments and inhibition tests, and considering the growth rates in Half Fraser and Fraser broths, the new species do not seem to interfere with the detection of L. monocytogenes.