Antimicrobial resistance and prevalence of virulence factor genes in fecal Escherichia coli of Holstein calves fed milk with and without antimicrobials

Diarrhea in calves has a significant effect on the dairy industry. A common management practice for preventing or decreasing the effects of such disease in preweaned calves is by the use of antimicrobials in milk or milk replacer. In this study, Escherichia coli antimicrobial resistance in fecal samples collected from calves 2 to 8 d of age that had or had not received antimicrobials in the milk and that had or had not signs of diarrhea by inspection of fecal consistency were investigated. Specifically, resistance of E. coli isolates to individual antimicrobials, multiresistance patterns, and presence of virulence factors were analyzed. Escherichia coli isolates were tested for susceptibility to 12 antimicrobials by use of a Kirby-Bauer disk diffusion assay. The study was conducted at 3 farms, 1 administering growth-promoting antimicrobials (GPA) in the milk and 2 not using GPA in the milk (NGPA). All isolates were susceptible to ciprofloxacin and cefepime. From the total isolates tested, 84% (n = 251) were resistant to at least 2 antimicrobials and 81% (n = 251) were resistant to 3 or more antimicrobials. When antimicrobial resistance was compared between GPA and NGPA, it was observed that the GPA group had higher odds of antimicrobial resistance for most of the individual antimicrobials tested. No significant correlation of virulence factors in GPA or NGPA and diarrheic or non-diarrheic (control) fecal samples was found. Of the 32 virulence factors evaluated, 21 were detected in the study population; the incidence of only 1 virulence factor was statistically significant in each of the diarrheic status (diarrheic or non-diarrheic) and treatment status (NGPA or GPA) groups. Phylogenetic analysis based on the nucleotide sequence of the DNA gyrase gene (gyrB) from 31 fecal E. coli isolates revealed 3 main clades.     

Herd-level relationship between antimicrobial use and presence or absence of antimicrobial resistance in gram-negative bovine mastitis pathogens on Canadian dairy farms

Concurrent data on antimicrobial use (AMU) and resistance are needed to contain antimicrobial resistance (AMR) in bacteria. The present study examined a herd-level association between AMU and AMR in Escherichia coli (n = 394) and Klebsiella species (n = 139) isolated from bovine intramammary infections and mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Antimicrobial use data were collected using inventory of empty antimicrobial containers and antimicrobial drug use rate was calculated to quantify herd-level AMU. Minimum inhibitory concentrations (MIC) were determined using Sensititre National Antimicrobial Resistance Monitoring System (NARMS) gram-negative MIC plate (Trek Diagnostic Systems Inc., Cleveland, OH). Isolates were classified as susceptible, intermediate, or resistant. Intermediate and resistant category isolates were combined to form an AMR category, and multivariable logistic regression models were built to determine herd-level odds of AMR to tetracycline, ampicillin, cefoxitin, chloramphenicol, trimethoprim-sulfamethoxazole combination, sulfisoxazole, streptomycin and kanamycin in E. coli isolates. In the case of Klebsiella species isolates, logistic regression models were built for tetracycline and sulfisoxazole; however, no associations between AMU and AMR in Klebsiella species were observed. Ampicillin-intermediate or -resistant E. coli isolates were associated with herds that used intramammarily administered cloxacillin, penicillin-novobiocin combination, and cephapirin used for dry cow therapy [odds ratios (OR) = 26, 32, and 189, respectively], and intramammary ceftiofur administered for lactating cow therapy and systemically administered penicillin (OR = 162 and 2.7, respectively). Use of systemically administered penicillin on a dairy farm was associated with tetracycline and streptomycin-intermediate or -resistant E. coli isolates (OR = 5.6 and 2.8, respectively). Use of cephapirin and cloxacillin administered intramammarily for dry cow therapy was associated with increasing odds of having at least 1 kanamycin-intermediate or -resistant E. coli isolate at a farm (OR = 8.7 and 9.3, respectively). Use of systemically administered tetracycline and ceftiofur was associated with cefoxitin-intermediate or -resistant E. coli (OR = 0.13 and 0.16, respectively); however, the odds of a dairy herd having at least 1 cefoxitin-intermediate or -resistant E. coli isolate due to systemically administered ceftiofur increased with increasing average herd parity (OR = 3.1). Association between herd-level AMU and AMR in bovine mastitis coliforms was observed for certain antimicrobials. Differences in AMR between different barn types and geographical regions were not observed.     

Prevalence and antimicrobial resistance of Salmonella in retail foods in northern China

A total of 387 retail meat, seafood and milk powder samples were collected from nine cities in northern China in 2005 and screened for the presence of Salmonella. Salmonella strains isolated were subjected to serotyping and antimicrobial susceptibility testing. Salmonella was isolated from 81 (20.9%, 81/387) samples and classified into 23 serotypes. The isolates were frequently resistant to sulfamethoxazole (86.4%), sulfamethoxazole/trimethoprim (48.1%), nalidixic acid (30.9%), tetracycline (19.8%), carboxybenzylpenicillin (17.3%), amoxicillin (17.3%) and ampicillin (16.0%). The multiple resistance (resistance to ≥ 3 antibiotics) was found in 29.6% (n = 24) isolates. Additionally, 4 isolates from chicken displayed the ACSSuTNx profile, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline and nalidixic acid, in particular, strain HBS084 showing the resistance to as many as 20 antibiotics. Salmonella from chicken showed the higher frequency of antimicrobial resistance. Our findings indicate that in northern China food products of animal origin can be a source of exposure for consumers to multiresistant Salmonella strains.     

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part II: In vitro antimicrobial activity evaluation of a bacteriophage cocktail and several antibiotics

The use of pathogenic-specific antimicrobials, as proposed by bacteriophage therapy, is expected to reduce the incidence of resistance development. Eighty Escherichia coli isolated from uteri of Holstein dairy cows were phenotypically characterized for antimicrobial resistance to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline by broth microdilution method. The lytic activity of a bacteriophage cocktail against all isolates was performed by a similar method. Additionally, the effect of different concentrations of antimicrobials and multiplicities of infections (MOI) of the bacteriophage cocktail on E. coli growth curve was measured. Isolates exhibited resistance to ampicillin (33.7%), ceftiofur (1.2%), chloramphenicol (100%), and florfenicol (100%). All strains were resistant to at least 2 of the antimicrobial agents tested; multidrug resistance (≥3 of 7 antimicrobials tested) was observed in 35% of E. coli isolates. The major multidrug resistance profile was found for ampicillin-chloramphenicol-florfenicol, which was observed in more than 96.4% of the multidrug-resistant isolates. The bacteriophage cocktail preparation showed strong antimicrobial activity against multidrug-resistant E. coli. Multiplicity of infection as low as 10⁻⁴ affected the growth of the E. coli isolates. The ratio of 10 bacteriophage particles per bacterial cell (MOI = 10¹) was efficient in inhibiting at least 50% of all isolates. Higher MOI should be tested in future in vitro studies to establish ratios that completely inhibit bacterial growth during longer periods. All isolates resistant to florfenicol were resistant to chloramphenicol and, because florfenicol was recently introduced into veterinary clinics, this finding suggests that the selection pressure of chloramphenicol, as well as other antimicrobials, may still play a relevant role in the emergence and dissemination of florfenicol resistance in E. coli. The bacteriophage cocktail had a notable capacity to inhibit the in vitro growth of E. coli isolates, and it may be an attractive alternative to conventional treatment of metritis by reducing E. coli in uteri of postpartum dairy cows.     

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in Gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7 log cfu/mL) for tetB and 5 × 10² cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r = 0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r = 0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.     

Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis

To evaluate the antimicrobial resistance traits of staphylococci responsible for subclinical bovine mastitis in Portugal, the minimum inhibitory concentrations (MIC) of 7 antimicrobial agents, frequently administered for mastitis treatment, were determined for 30 Staphylococcus aureus and 31 Staphylococcus epidermidis field isolates. β-Lactamase production was detected through the use of nitrocefin-impregnated discs. The MIC that inhibited 90% of the isolates tested (MIC₉₀) of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were, respectively, 4, 0.5, 1, 1, 0.25, 0.25, and 0.06 μg/mL for Staph. aureus and ≥64, 8, 1, 32, ≥64, ≥64, and 0.06 μg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. β-Lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were β-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 μg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.     

Identification, susceptibility, and detection of integron-gene cassettes of Arcanobacterium pyogenes in bovine endometritis

The present study aimed to identify, determine the susceptibility, and detect gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis. Arcanobacterium pyogenes isolates were identified first by using the API Coryne Vit system test, and further through PCR. Minimum inhibitory concentrations of 23 antimicrobial agents against A. pyogenes were tested using standard broth microdilution assays according to the protocols of the Clinical and Laboratory Standards Institute. The genes of integrons I and II were amplified by PCR using specific primers. Thirty-two A. pyogenes isolates were isolated from 136 endometritic cows in the Hohhot region. Antibiotic susceptibility tests revealed that all isolates were highly sensitive to fluoroquinolones (100%), macrolides (∼81.2 to 100%) and florfenicol (90.6%), aminoglycosides (∼15.6 to 81.2%), and tetracyclines (∼43.7 to 68.7%). However, 53.1% were resistant to clindamycin, ∼50 to 65.6% were resistant to penicillins, and ∼37.5 to 71.9% were resistant to cephalosporins. One hundred percent were resistant to sulfonamides and bacitracin zinc. The integrons were further confirmed by sequencing. No class II integrons were detected, whereas 50% (n = 16) of the A. pyogenes isolates were positive for the presence of the intI I gene, but only 13 contained gene cassettes. Sequence analysis of gene cassettes revealed 6 gene cassettes, 4 of which encode resistant determinants of aminoglycosides (aadA1, aadA5, aadA24, and aadB) and 1 of which encodes the resistance gene of chloramphenicol (cmlA6). The function of the sixth identified cassette, designated ORF1, is unknown. The gene cassette arrays aadA24-ORF1, aadA5, and aadA1-addB-cmlA6 were found in 46.13% (6/13), 38.46% (5/13), and 38.46% (5/13) of the isolates, respectively. These cassettes segregated according to a consistent pattern, with aadA5 always alone, ORF1 always with aadA24, and aadA1-aadB and cmlA6 always together. Most of the positive integrons existed in the multiresistant isolates (n = ∼3 to 7), indicating that the integrons played an important role in the dissemination and spread of antimicrobial resistance. This is the first report of A. pyogenes infections in dairy cows in China and of detection of gene cassettes and integrons in A. pyogenes.     

Short communication: Genetic characterization of antimicrobial resistance in Acinetobacter isolates recovered from bulk tank milk

A total of 176 Acinetobacter isolates, including 57 Acinetobacter baumannii originally obtained from 2,287 bulk tank milk (BTM) samples in Korea was investigated for the genetic basis of antimicrobial resistance using molecular methods. In addition, the occurrence and cassette content of integrons were examined and the genetic diversity of A. baumannii strains identified was evaluated. Aminoglycoside-modifying enzyme genes were detected in 15 (88.2%) of the 17 aminoglycoside-resistant Acinetobacter isolates tested. The most common aminoglycoside-modifying enzyme gene identified was adenylyltransferase gene aadB (n = 9), followed by phosphotransferase genes aphA6 (n = 7) and aphA1 (n = 5). Of the 31 isolates resistant to tetracycline, tet(39) was detected in 20 of them. The genetic basis of resistance to sulfonamide was identified in 15 (53.6%) of 28 trimethoprim-sulfamethoxazole-resistant isolates and 9 (32.1%) of them carried both sul1 and sul2 genes. A bla_ADC-7-like gene was detected in 1 β-lactam-resistant A. baumannii. Furthermore, class 1 integron was identified in 11 Acinetobacter isolates. Two gene cassettes dfrA15, conferring resistance to trimethoprim, and aadA2, conferring resistance to aminoglycosides, were identified in 8 Acinetobacter isolates. None of the isolates was positive for class 2 or class 3 integrons. Pulsed-field gel electrophoresis revealed that most of the A. baumannii strains from BTM samples were genetically diverse, indicating that the occurrence of A. baumannii strains in BTM was not the result of dissemination of a single clone. Elucidation of resistance mechanisms associated with the resistance phenotype and a better understanding of resistance genes may help in the development of strategies to control infections, such as mastitis, and to prevent further dissemination of antibiotic resistance genes. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial-resistant Acinetobacter spp. from milk.     

Inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms

We investigated the potential use of biofilm formed by a competitive-exclusion (CE) microorganism to inactivate Escherichia coli O157:H7 on a stainless steel surface. Five microorganisms showing inhibitory activities against E. coli O157:H7 were isolated from vegetable seeds and sprouts. The microorganism with the greatest antimicrobial activity was identified as Paenibacillus polymyxa (strain T5). In tryptic soy broth (TSB), strain T5 reached a higher population at 25 °C than at 12 or 37 °C without losing inhibitory activity against E. coli O157:H7. When P. polymyxa (6 log CFU/mL) was co-cultured with E. coli O157:H7 (2, 3, 4, or 5 log CFU/mL) in TSB at 25 °C, the number of E. coli O157:H7 decreased significantly within 24 h. P. polymyxa formed a biofilm on stainless steel coupons (SSCs) in TSB at 25 °C within 24 h, and cells in biofilms, compared to attached cells without biofilm formation, showed significantly increased resistance to a dry environment (43% relative humidity [RH]). With the exception of an inoculum of 4 log CFU/coupon at 100% RH, upon exposure to biofilm formed by P. polymyxa on SSCs, populations of E. coli O157:H7 (2, 4, or 6 log CFU/coupon) were significantly reduced within 48 h. Most notably, when E. coli O157:H7 at 2 log CFU/coupon was applied to SSCs on which P. polymyxa biofilm had formed, it was inactivated within 1 h, regardless of RH. These results will be useful when developing strategies using biofilms produced by competitive exclusion microorganisms to inactivate foodborne pathogens in food processing environments.     

Application of an active alginate coating to control the growth of Listeria monocytogenes on poached and deli turkey products

The relatively high prevalence of Listeria monocytogenes in ready-to-eat (RTE) turkey products is of great concern. The overall objective of this study was to develop antimicrobial edible coating formulations to effectively control the growth of this pathogen. The antimicrobials studied were nisin (500 IU/g), Novagard CB 1 (0.25%), Guardian NR100 (500 ppm), sodium lactate (SL, 2.4%), sodium diacetate (SD, 0.25%), and potassium sorbate (PS, 0.3%). These were incorporated alone or in binary combinations into five edible coatings: alginate, κ-carrageenan, pectin, xanthan gum, and starch. The coatings were applied onto the surface of home-style poached and processed deli turkey discs inoculated with ~ 3 log CFU/g of L. monocytogenes. The turkey samples were then stored at 22 °C for 7 days. For poached and processed deli turkey, the coatings were found to be equally effective, with pectin being slightly less effective than the others. The most effective poached turkey treatments seemed to be SL (2.4%)/SD (0.25%) and Nisin (500 IU/g)/SL (2.4%), which yielded final populations of 3.0 and 4.9 log CFU/g respectively compared to the control which was 7.9 log CFU/g. For processed deli turkey, the most effective antimicrobial treatments seemed to be Nisin (500 IU/g)/SD (0.25%) and Nisin (500 IU/g)/SL (2.4%) with final populations of 1.5 and 1.7 log CFU/g respectively compared to the control which was 6.5 log CFU/g. In the second phase of the study, home-style poached and store-purchased roasted (deli) turkey inoculated with the pathogen at a level of ~ 3 log CFU/g were coated with alginate incorporating selected antimicrobial combinations and stored for 8 weeks at 4 °C. Alginate coatings supplemented with SL (2.4%)/PS (0.3%) delayed the growth of L. monocytogenes with final counts reaching 4.3 log CFU/g (home-style poached turkey) and 6.5 log CFU/g (roasted deli turkey) respectively while the counts in their untreated counterparts were significantly higher (P < 0.05) reaching 9.9 and 7.9 log CFU/g, respectively. This study therefore demonstrates the effectiveness of using alginate-based antimicrobial coatings to enhance the microbiological safety and quality of RTE poultry products during chilled storage.     

Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities

The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n = 11), other bacterial pathogenic and spoilage bacteria (n = 7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log₁₀ cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p > 0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p > 0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥ 2 mg/ml did not significantly (p > 0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required.     

Role of O-antigen on the Escherichia coli O157:H7 cells hydrophobicity, charge and ability to attach to lettuce

Environmental factors encountered during growing and harvesting may contribute to Escherichia coli O157:H7 contamination of lettuce. Limited nutrients and extended exposure to water may cause E. coli O157:H7 to shed its O antigen. Absence of the O157-polysaccharide antigen could affect the cell's physicochemical properties (hydrophobicity and cell charge) and ultimately influence its attachment to surfaces. The objectives of this study were to evaluate the effect of the E. coli O157:H7 O-antigen on the cell's overall hydrophobicity, charge and ability to attach to cut edge and whole leaf iceberg lettuce surfaces. Three strains of E. coli O157:H7 (86-24 wild type; F-12, mutant lacking the O-antigen and pRFBE, plasmid for O157 gene reintroduced) were examined for their hydrophobicity, overall charge and ability to attach to lettuce. Overall, E. coli O157:H7 attached at higher levels to cut surfaces over whole leaf surfaces (P = 0.008) for all strains and treatments. Additionally, the strain lacking the O-antigen (F12) — attached significantly less to lettuce (P = 0.015) than the strains expressing the antigen (WT and pRFBE). Cells lacking the O antigen (strain F-12) were also significantly more hydrophobic than strains 86-24 or pRFBE (P ≤ 0.05). Surface charge differed among the strains tested (P ≤ 0.05); however, it did not appear to influence bacterial attachment to lettuce surfaces. The charge was not fully restored in the pRFBE strain (expression of O-antigen reintroduced), therefore, no conclusions can be made pertaining to the effect of charge on attachment in this study. Results indicate that E. coli O157:H7 cells which lack the O-antigen have greater hydrophobicity and attach at lower concentrations than cells expressing the O-antigen, to iceberg lettuce surfaces.     

Microbial,instrumental color and sensory characteristics of inoculated ground beef produced using potassium lactate,sodium metasilicate or peroxyacetic acid as multiple antimicrobial interventions

Effectiveness of multiple antimicrobial interventions on ground beef microbial, instrumental color and sensory attributes through display was evaluated. Beef trimmings were inoculated with Escherichia coli (EC) and Salmonella typhimurium (ST) then treated with either: (1) 3% potassium lactate followed by 4% sodium metasilicate (KN); (2) 4% sodium metasilicate followed by 3% potassium lactate (NK); (3) 200-ppm peroxyacetic acid followed by 3% potassium lactate (PK); (4) 200-ppm peroxyacetic acid followed by 4% sodium metasilicate (PN); or control (CON). Trimmings were ground, packaged and sampled on days 0–7 of display for EC, ST, coliforms, aerobic plate count, instrumental color and sensory characteristics. Only PK reduced (P < 0.05) all bacterial types evaluated. The PN treatment remained (P < 0.05) redder (a*), contained more (P < 0.05) oxymyoglobin and had less (P < 0.05) discoloration than CON by days 3–7 of display. All treatments maintained or improved odor attributes.     

Escherichia coli of poultry food origin as reservoir of sulphonamide resistance genes and integrons

The antimicrobial resistance phenotype and genotype, the flanking regions of sulphonamide resistance genes and the integrons were analyzed in 166 Escherichia coli isolates recovered from poultry meat in Tunisia. High percentages of resistance were detected to ampicillin, streptomycin, nalidixic acid, sulphonamide and tetracycline (66-95%), and lower percentages to gentamicin, amoxicillin-clavulanic acid and cefoxitin (1-4%). The bla_TEM, tet(A)/tet(B), aph(3′)-Ia, aac(6′)-Ib-cr, aac(3)-II and cmlA genes were identified in 92, 82, 29, 2, 2 and 7 isolates, respectively. Class 1 and/or class 2 integrons were detected in 52% of E. coli isolates and five different gene cassette arrangements were identified in the variable regions of class 1 integrons, which included antimicrobial resistance determinants. Sixty-eight isolates contained the sul1 gene and 37 of them presented this gene into a class 1 integron structure. The sul3 gene was detected associated with non-classic class 1 integrons in 4 out of 46 sul3-positive isolates. The sul2 gene was detected in 66 isolates, 51 of them were linked to strA/B genes in seven different genetic structures. Seventy-three-per-cent of integron-positive isolates presented resistance to at least five different antimicrobial families versus 38.7% of integron-negative isolates. Our study highlights the role of commensal E. coli isolates from poultry meat as an important reservoir for sulphonamide resistance genes and integrons carrying antimicrobial resistance genes.     

A survey of oysters (Crassostrea gigas) in New Zealand for Vibrio parahaemolyticus and Vibrio vulnificus

A microbiological survey was conducted to determine the levels of total and pathogenic Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) in Pacific oysters (Crassostrea gigas) collected from commercial growing areas in the North Island, New Zealand.The survey was intended to be geographically representative of commercial growing areas of Pacific oysters in New Zealand, while selecting the time frame most likely to coincide with the increased abundance of pathogenic vibrio species. Vp was detected in 94.8% of oyster samples examined (n = 58) with a geometric mean concentration of 99.3 MPN/g, while Vv was detected in 17.2% of oyster samples examined with a geometric mean concentration of 7.4 MPN/g. The frequency of Vp positive samples was 1.7 fold greater than reported in a study conducted three decades ago in New Zealand. Potentially virulent (tdh positive) Vp was detected in two samples (3.4%, n = 58) while no trh (another virulence marker) positive samples were detected. 16 S rRNA genotype could be assigned only to 58.8% of Vv isolates (8:1:1 A:B:AB ratio, n = 10). There was a good agreement [98.2% of Vp (n = 280) and 94.4% of Vv (n = 18) isolates] between molecular tests and cultivation based techniques used to identify Vibrio isolates and there was a significant (R² = 0.95, P < 0.001, n = 18) linear relationship between the MPN estimates by real-time PCR and cultivation. There was no significant correlation between any of the environmental parameters tested and Vp or Vv concentrations.     

Microbiological and organoleptic characteristics of beef trim and ground beef treated with acetic acid, lactic acid, acidified sodium chlorite, or sterile water in a simulated commercial processing environment to reduce Escherichia coli O157:H7 and Salmonella

The objective of this study was to validate the effectiveness of acetic and lactic acids (2% and 5%), acidified sodium chlorite (1000 ppm), and sterile water in reducing Escherichia coli O157:H7 and Salmonella Typhimurium in inoculated beef trim in a simulated processing environment. Samples were collected to assess microbial characteristics at three processing points. Results from this study indicate that all treatments, including sterile water, reduced pathogen concentrations (P < 0.05) of both E. coli O157:H7 and Salmonella Typhimurium in ground beef up to 0.5 and 0.6 log by 24 h, respectively. In some cases, there were no significant differences between the antimicrobial treatments and the sterile water using this application method. Triangle sensory test results of non-inoculated beef indicated there were no differences (P < 0.05) in the means of correct responses between controls or antimicrobial treatments at 6 or 24 h. While interventions are important for beef trim, use of the interventions must be validated under industry conditions to ensure proper effectiveness.     

Characterization of bacterial susceptibility isolates in sixteen dairy farms in Taiwan

Bacteria were isolated from dairy cows, dairy farm environments, and dairy workers in 2 geographically different areas of eastern and northern Taiwan. Isolates were evaluated for antimicrobial susceptibility and the phylogenetics of isolated Escherichia coli O157:H7 were characterized. A total of 1,346 bacteria were identified, including 226 E. coli, 30 Pseudomonas spp. (7 Pseudomonas aeruginosa), 259 other gram-negative bacteria, 271 Enterococcus spp., 314 Staphylococcus spp., 195 Streptococcus spp., and 51 other gram-positive bacteria. Among them, 88% (1,184/1,346) of the isolates were resistant to sulfadimethoxine. The percentages of gram-negative bacteria resistant to oxy-tetracycline and streptomycin were 48% (249/515) and 78% (404/515), respectively. Gram-positive bacteria isolated from eastern Taiwan, the least polluted region of Taiwan, were found to have greater antimicrobial resistance than those isolated from northern Taiwan. Two E. coli O157:H7 from 2 different geographical areas were isolated. Both were vt2-positive but vt1-negative and had phylogenetic similarities of 82 and 67%, respectively, compared with previous isolates. Information on antimicrobial susceptibility revealed from this dairy farm survey may serve as a baseline for future studies and may also highlight the need to formulate better regulation strategies for the safe use of antimicrobials on food-producing farms.     

Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione, fludioxonil and cyprodinil and characterization of fitness parameters and patulin production

A total of 236 Penicillium expansum field isolates from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, Northern Greece were tested for their sensitivity to tebuconazole, fludioxonil, iprodione and cyprodinil. Preliminary fungitoxicity tests on the response of the isolates showed several phenotypes, distinguished according to their sensitivity to fungicides tested. The EC₅₀ values ranged from 0.64 to 5 (average = 0.98) μg/ml for iprodione, 0.9 to 7.3 (average = 2.66) μg/ml for tebuconazole, 0.008 to 1.28 (average = 0.55) μg/ml for cyprodinil and from 0.013 to 0.47 (average = 0.08) μg/ml for fludioxonil. A bimodal distribution of the EC₅₀ values of isolates with distinct sensitive and resistant populations to fludioxonil and tebuconazole were observed. In the case of cyprodinil, a much broader, hundred-fold, range of sensitivity was found, probably indicating that some isolates are relatively insensitive to cyprodinil compared to the most sensitive ones. Isolates exhibiting simultaneously reduced sensitivity to tebuconazole and fludioxonil or tebuconazole and iprodione or to tebuconazole and cyprodinil were also observed at low frequencies. A small portion of the population (7.5%) showed multiple resistance to tebuconazole, fludioxonil and iprodione. Study of fitness determining parameters showed that the resistance to tebuconazole, fludioxonil and iprodione had a significant adverse effect on mycelial growth rate and pathogenicity. Contrary to that, these fitness parameters were not affected in the isolates showing reduced sensitivity to cyprodinil. Analysis of patulin production on YES-agar growth medium and on artificially inoculated apple fruit showed that all isolates were mycotoxigenic. Most of the cyprodinil-insensitive isolates produced patulin at concentrations similar to or relatively higher (up to 1.5-fold on growth medium) than the sensitive ones. In contrast, a significant reduction (up to 98% of multiple resistant isolates) in patulin production was observed in all other phenotypes, indicating an adverse effect of fitness penalties on the mycotoxigenic ability of resistant isolates. The above mentioned data clearly show a considerable risk for the selection of P. expansum isolates resistant to fludioxonil, iprodione, tebuconazole and cyprodinil. The potential risk of increased patulin contamination of apples and their byproducts by the appearance and predominance of highly mycotoxigenic isolates of P. expansum resistant to the anilinopyrimidines is discussed.     

Application of caffeine, 1,3,7-trimethylxanthine,to control Escherichia coli O157:H7

The objective of this research was to determine the effectiveness of caffeine on inactivation of Escherichia coli O157:H7 in brain heart infusion (BHI) broth. Overnight samples of five E. coli O157:H7 strains of (E0019, F4546, H1730, 944 and Cider) were used in this study. These strains were individually inoculated at an initial inoculum level of 2 log CFU/ml into BHI broth containing caffeine at different concentrations (0.00%, 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, and 2.00%). Samples were then incubated at 37 °C for 24 h. Bacterial growth was monitored at different time intervals by measuring turbidity at 610 nm using a spectrophotometer. Results revealed that the addition of caffeine inhibited the growth of E. coli O157:H7. Significant growth inhibition was observed with concentration levels of 0.50% and higher. These results indicate that caffeine has potential as an antimicrobial agent for the treatment of E. coli O157:H7 infection and should be investigated further as a food additive to increase biosafety of consumable food products.     

Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde

The effect of trans-cinnamaldehyde (TC) on the inactivation of Escherichia coli O157:H7 in undercooked ground beef patties was investigated. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (7.0 log CFU/g), followed by addition of TC (0, 0.15, and 0.3%). The meat was formed into patties and stored at 4 °C for 5 days or at −18 °C for 7 days. The patties were cooked to an internal temperature of 60 or 65 °C, and E. coli O157:H7 was enumerated. The numbers of E. coli O157:H7 did not decline during storage of patties. However, cooking of patties containing TC significantly reduced (P < 0.05) E. coli O157:H7 counts, by >5.0 log CFU/g, relative to the reduction in controls cooked to the same temperatures. The D-values at 60 and 65 °C of E. coli O157:H7 in TC-treated patties (1.85 and 0.08 min, respectively) were significantly lower (P < 0.05) than the corresponding D-values for the organism in control patties (2.70 and 0.29 min, respectively). TC-treated patties were more color stable and showed significantly lower lipid oxidation (P < 0.05) than control samples. TC enhanced the heat sensitivity of E. coli O157:H7 and could potentially be used as an antimicrobial for ensuring pathogen inactivation in undercooked patties. However detailed sensory studies will be necessary to determine the acceptability to consumers of TC in ground beef patties.