Thermal inactivation D- and z-values of multidrug-resistant and non-multidrug-resistant Salmonella serotypes and survival in ground beef exposed to consumer-style cooking.

There has been speculation that multidrug-resistant (MDR) strains are generated by subtherapeutic antibiotic use in food animals and that such strains result in increased resistance to lethality by food processes such as heat and irradiation. The objective of this study was to evaluate the heat resistance of 20 strains, namely an MDR and a non-multidrug-resistant (NMDR) strain of each of 10 Salmonella serotypes isolated from cattle or cattle environments. MDR and NMDR Salmonella serotypes studied included Montevideo, Typhimurium, Anatum, Muenster, Newport, Mbandaka, Dublin, Reading, Agona, and Give. For phase I, stationary-phase cultures of the strains were aliquoted into sterile capillary tubes and immersed in a temperature-controlled water bath at 55, 60, 65, and 70 degrees C for appropriate times. Survivor curves were plotted for each temperature, and a best-fit linear regression was derived for each temperature. D-values (decimal reduction times) and z-values (changes in temperature required to change the D-values) were calculated for each strain. Although there was no overall significant difference in the heat resistance of MDR and NMDR serotypes, NMDR serotypes generally appeared to have slightly higher heat resistance than NMDR serotypes, especially at 55 and 60 degrees C. The highest relative heat resistance (highest z-values) was exhibited by Salmonella Anatum. Notably, the relative heat resistance of NMDR Salmonella Agona was similar to that of NMDR Salmonella Anatum and had the highest D-values at all four temperatures. For phase II, three serotypes (regardless of resistance profile) with the highest relative heat resistance and their drug-resistant counterparts were selected for thermal inactivation in ground beef patties cooked to endpoint temperatures. Salmonella Agona was able to survive in ground beef cooked to an internal temperature of 71 degrees C. Results of these studies suggest drug resistance does not affect the heat resistance of Salmonella and that serotype or strain is an important consideration in risk assessment of the pathogen with regard to survival at cooking temperatures.

Validation of individual and multiple-sequential interventions for reduction of microbial populations during processing of poultry carcasses and parts.

Changes in aerobic plate counts (APC), total coliform counts (TCC), Escherichia coli counts (ECC), and Salmonella incidence on poultry carcasses and parts and in poultry processing water were evaluated. Bacterial counts were estimated before and after individual interventions and after poultry carcasses were exposed to multiple-sequential interventions at various stages during the slaughter process. Individual and multiple-sequential interventions were evaluated at three processing plants: (i) plant A (New York wash, postevisceration wash, inside-outside bird washes 1 and 2, chlorine dioxide wash, chlorine dioxide wash plus chlorine chiller, chiller exit spray, and postchiller wash), (ii) plant B (New York wash, inside-outside bird washes 1 and 2, trisodium phosphate wash, and chlorine chiller), and (iii) plant C (trisodium phosphate wash and chlorine chiller). The majority of individual interventions effectively or significantly (P < 0.05) reduced microbial populations on or in carcasses, carcass parts, and processing water. Reductions in APC, TCC, and ECC due to individual interventions ranged from 0 to 1.2, 0 to 1.2, and 0 to 0.8 log CFU/ml, respectively. Individual interventions reduced Salmonella incidence by 0 to 100% depending on the type of process and product. Multiple-sequential interventions resulted in significant reductions (P < 0.05) in APC, TCC, ECC, and Salmonella incidence of 2.4, 2.8, and 2.9 log CFU/ml and 79%, respectively, at plant A; 1.8, 1.7, and 1.6 log CFU/ml and 91%, respectively, at plant B; and 0.8, 1.1, and 0.9 log CFU/ml and 40%, respectively, at plant C. These results enabled validation of in-plant poultry processing interventions and provide a source of information to help the industry in its selection of antimicrobial strategies.

Nationwide microbiological baseline data collected by sponge sampling during 1997 and 1998 for cattle, swine, turkeys, and geese.

During 1997 and 1998, the U.S. Food Safety and Inspection Service completed nationwide microbiological baseline studies on four separate categories of livestock and poultry. Data were collected by sponge-sampling techniques. These studies were designed to provide nationwide estimates of the prevalence of Salmonella and prevalence and concentrations of Escherichia coli in cattle (n = 1,881), swine (n = 2,127), turkeys (n = 1,396), and geese (n = 102) in establishments under federal inspection. Salmonella prevalence ranged from 1.2% in cattle to 6.9% in swine, 13.7% in geese, and 19.6% in turkeys. The prevalence of E. coli was 16.6% in cattle (geometric mean = 0.26 CFU/cm2), 44.1% in swine (mean = 0.78 CFU/cm2), 92.7% in turkey (mean = 2.46 CFU/cm2), and 96.5% in geese (mean = 1.97 CFU/cm2). These values are similar to or somewhat lower than previous baseline values obtained for steers and heifers, cows and bulls, market hogs, and young turkeys. This study is the first in which nationwide microbiological baseline data have been compiled for geese. These data will be useful to individuals working with hazard analysis critical control point plans and risk assessment and to the research and academic communities.

Coordination chemistry of silver(I) with the nitrogen-bridged ligands (C(6)H(5))(2)PN(H)P(C(6)H(5))(2) and (C(6)H(5))(2)PN(CH(3))P(C(6)H(5))(2): the effect of alkylating the nitrogen bridge on ligand bridging versus chelating behavior.

The coordination chemistry of silver(I) with the nitrogen-bridged ligands (C(6)H(5))(2)PN(R)P(C(6)H(5))(2) [R = H (dppa); R = CH(3) (dppma)] has been investigated by (31)P NMR and electrospray mass spectrometry (ESMS). Species observed by (31)P NMR include Ag(2)(mu-dppa)(2+), Ag(2)(mu-dppa)(2)(2+), Ag(2)(mu-dppa)(3)(2+), Ag(2)(mu-dppma)(2+), Ag(2)(mu-dppma)(2)(2+), and Ag(eta(2)-dppma)(2)(+). Species observed by ESMS at low cone voltages were Ag(2)(dppa)(2)(2+), Ag(2)(dppa)(3)(2+), Ag(2)(dppma)(2)(2+), and Ag(dppma)(2)(+). (C(6)H(5))(2)PN(CH(3))P(C(6)H(5))(2) showed a strong tendency to chelate, while (C(6)H(5))(2)PN(H)P(C(6)H(5))(2) preferred to bridge. Differences in the bridging versus chelating behavior of the ligands are assigned to the Thorpe-Ingold effect, where the methyl group on nitrogen sterically interacts with the phenyl groups on phosphorus. The crystal structure of the three-coordinate dinuclear silver(I) complex (Ag(2)[(C(6)H(5))(2)PN(H)P(C(6)H(5))(2)](3))(BF(4))(2) has been determined. Bond distances include Ag-Ag = 2.812(1) A, Ag(1)-P(av) = 2.492(3) A, and Ag(2)-P(av) = 2.509(3) A. The compound crystallizes in the monoclinic space group Cc at 294 K, with a = 18.102(4)(o), Z = 4, V = 7261(3) A(3), R = 0.0503, and R(W) = 0.0670.

Listeria monocytogenes lineage group classification by MAMA-PCR of the listeriolysin gene.

Nucleotide sequence differences within several virulence genes, including the listeriolysin O (hly) gene, are associated with three evolutionary lineage groups of Listeria monocytogenes. Because the ability of L. monocytogenes to cause disease may vary by evolutionary lineage group, rapid discrimination among the three lineage types may be important for estimating pathogenic potential. A Mismatch Amplification Mutation Assay (MAMA) was developed and used to rapidly screen and characterize L. monocytogenes isolates with regard to lineage type. A standard PCR amplified a 446-bp region within the hly gene with all three L. monocytogenes lineage genotypes. MAMA primers to four different sites within this region of the hly gene were designed to amplify under the same PCR conditions and generated amplicons, the size of which depended on the isolate genotype. Ninety-seven L. monocytogenes isolates were screened. All isolates, except ATCC 19116, could be classified by MAMA PCR as one of the three hly genotypes. Overall, 56, 36, and 4 of the 97 isolates tested were type 1, 2, or 3 respectively. Among the 26 patient isolates, 85%, 15%, and 0% were type 1, 2, or 3 respectively; for the 60 food isolates, 54% were type 1, 43% were type 2, and 3% were type 3. The combination of these MAMA PCR analyses provides a rapid method to screen and categorize L. monocytogenes isolates because of conserved nucleotide differences within the hly gene.

Phenanthroline-Cu(II) cleavage as a probe of rRNA structure.

Chemical cleavage is developing into a powerful tool for analysis and characterization of nucleic acids. Phenanthroline-Cu(II) cleavage has been used extensively for studies of DNA for the last two decades, but recently has been applied to structural studies of RNA as well. This approach has been used to study the structure and structural changes occurring in ribosomal RNA within the ribosomes. In this article we discuss the mechanism by which phenanthroline cleaves, the applications possible using this approach, and the results that can be obtained. Protocols for use of phenanthroline are outlined as well.

Comparison of rRNA cleavage by complementary 1,10-phenanthroline-Cu(II)- and EDTA-Fe(II)-derivatized oligonucleotides.

The chemical nucleases 1,10-phenanthroline-Cu(II) and EDTA-Fe(II), have proven to be valuable tools for structural analysis of nucleic acids. Both have found applications in footprinting and directed proximity studies of DNA and RNA. Derivatives of each that provide for tethering to nucleic acid or protein are commercially available, allowing their widespread use for structural analysis of macromolecules. Although their applications are somewhat overlapping, differences in their cleavage mechanisms and chemical properties allow them to provide distinct and complementary structural information. The purpose of this study is to compare directly the cleavage patterns of tethered 1,10-phenanthroline-Cu(II) and EDTA-Fe(II) complexes within a similar experimental system. Here, the region surrounding nucleotide 1400 of 16S rRNA from Escherichia coli serves as a substrate for chemical cleavage directed by a derivatized complementary oligonucleotide. This region of rRNA is known to be involved in the decoding of mRNA during translation. The results of this study provide evidence in support of the mechanistic differences previously established for EDTA-Fe(II) and 1,10-phenathroline-Cu(II). The delocalized cleavage envelope produced by EDTA-Fe(II) cleavage suggests the involvement of a diffusible reactive species. On the other hand, rRNA cleavage induced by the tethered 1,10-phenanthroline-Cu(II) complex appears localized to the proximity of the chemical nuclease under normal conditions, although the production of an unknown diffusible species appears to occur during long reaction times.

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit.

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus.

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.

Positions in the 30S ribosomal subunit proximal to the 790 loop as determined by phenanthroline cleavage.

Positioning rRNA within the ribosome remains a challenging problem. Such positioning is critical to understanding ribosome function, as various rRNA regions interact to form suitable binding sites for ligands, such as tRNA and mRNA. We have used phenanthroline, a chemical nuclease, as a proximity probe, to help elucidate the regions of rRNA that are near neighbors of the stem-loop structure centering at nt 790 in the 16S rRNA of the Escherichia coli 30S ribosomal subunit. Using phenanthroline covalently attached to a DNA oligomer complementary to nt 787-795, we found that nt 582-584, 693-694, 787-790, and 795-797 were cleaved robustly and must lie within about 15 A of the tethered site at the 5' end of the DNA oligomer, which is adjacent to nt 795 of 16S rRNA.

An oligonucleotide-ligation assay for the differentiation between Cyclospora and Eimeria spp. polymerase chain reaction amplification products.

An oligonucleotide-ligation assay (OLA) was developed and compared to a restriction fragment length polymorphism (RFLP) test for distinguishing between 294-bp polymerase chain reaction (PCR) amplification products of the 18S rRNA gene from Cyclospora and Eimeria spp. The PCR/OLA correctly distinguished between three Cyclospora, three E. tenella, and one E. mitis strains and the ratio of positive to negative spectrophotometric absorbance (A490) values for each strain ranged from 4.086 to 15.280 (median 9.5). PCR/OLA provides a rapid, reliable, spectrophotometric alternative to PCR/RFLP.

Development of digoxigenin-labeled PCR amplicon probes for use in the detection and identification of enteropathogenic Yersinia and Shiga toxin-producing Escherichia coli from foods.

By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), amplification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathogenic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. This technology allows the synthesis of a variety of shelf-stable probe reagents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: virF and yadA in enteropathogenic Yersinia and stx1 and stx2 in Shiga-like toxin-producing Escherichia coli. Results of DNA hybridizations of dot blots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent with results of equivalent PCR analyses. DNA colony hybridization with nonisotopic virF probes of colonies arising on spread plates from artificially contaminated food homogenates was able to detect potentially pathogenic Y. enterocolitica. When compared with oligonucleotide probes, amplicon probes are much less sensitive to changes in hybridization and wash temperatures, allowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at -20 degrees C for more than 8 months. This method conveniently generates probes that are safe, stable, inexpensive, reusable, and reliable.

Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II).

Studying the intricate folding of rRNA within the ribosome remains a complex problem. Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion. We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100. This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA. It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data. The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA.

Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus.

The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed. An alkaline phosphatase (AP)-labelled probe was evaluated for specificity against 26 strains of V. parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non-vibrio species. Of the 124 isolates tested, the probe hybridized only with the 26 strains of V. parahaemolyticus, indicating species specificity. Two hundred and six suspect V. parahaemolyticus isolates from oysters were tested by this probe and API-20E diagnostic strips; there was 97% agreement between results. A digoxigenin (DIG)-labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP-labelled probe. When tested on 584 suspect V. parahaemolyticus isolates, results obtained with the AP- and DIG-labelled probes were in 98% agreement. These results suggest that the probes are equivalent for detection of the V. parahaemolyticus tlh gene.

Template preparation for PCR and RFLP of amplification products for the detection and identification of Cyclospora sp. and Eimeria spp. Oocysts directly from raspberries.

Raspberries were epidemiologically associated with cyclosporiasis outbreaks during 1996 and 1997. The 18S rRNA genes of Cyclospora cayetanensis and several species of a closely related genus, Eimeria, were sequenced and primers for a nested PCR developed in a previous study. The ability to distinguish amplified products of Cyclospora sp. from those of Eimeria spp. is important for testing food and environmental samples. Therefore, an RFLP analysis of amplified products was used to differentiate Cyclospora cayetanensis from Eimeria spp. PCR inhibitors and the low levels of Cyclospora oocysts present in raspberries make template preparation for PCR challenging. Several approaches for PCR template preparation from raspberry samples were evaluated. Template preparation methods using various washing and concentration steps, oocyst disruption protocols, resin matrix treatment, DNA precipitation, and/or the addition of nonfat dried milk solution to a PCR using modified primers were evaluated first with oocysts of Eimeria tenella then refined with oocysts of C. cayetanensis. Approximately 10 E. tenella oocysts per PCR or approximately 19 C. cayetanensis oocysts per PCR were detected with the optimized template preparation method. The addition of 20 microliters of raspberry wash sediment extract and nonfat dried milk solution did not inhibit the amplification of DNA from as few as 10 E. tenella and 25 C. cayetanensis oocysts in a 100-microliter PCR. The nucleotide sequences of C. cayetanensis and the Eimeria spp. are 94 to 96% similar in the amplified region, but the amplification products from the two genera were distinguished using an RFLP analysis with the restriction enzyme MnlI.

Regions of 16S ribosomal RNA proximal to transfer RNA bound at the P-site of Escherichia coli ribosomes.

Unmodified uridines have been randomly replaced by 4-thiouridines in transfer RNAPhe (tRNAPhe) transcribed in a T7 RNA polymerase system. These 4-thiouridines serve as conjugation sites for attachment of the cleavage reagent 5-iodoacetamido-1,10-o-phenanthroline (IoP). In a reducing environment, when complexed with Cu2+, 1,10-o-phenanthroline causes cleavage of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-oP) conjugates, when bound at the P-site of 70S ribosomes and 30S ribosomal subunits, caused cleavage of ribosomal RNA (rRNA) mainly in domains I and II of 16S rRNA. Some positions were cleaved only when tRNA-oP was bound to 70S ribosomes or to 30S ribosomal subunits. In domain I, most cleavage sites occurred in or near the 530 pseudoknot region. In domain II, most nucleotides cleaved were near the 690 region and the 790 region. The only positions cleaved in domain III were near the 1050 region. There were no discernible nucleotides cleaved near the 1400 (decoding) region. Our results corroborated results of others, which have shown these sites to be protected from chemical modification by tRNA binding or to be cross-linked to P-site-bound tRNA. Use of cleavage reagents tethered to tRNA provides evidence for additional regions of rRNA that may be proximal to bound tRNA.

Comparative sensitivity of 13 species of pathogenic bacteria to seven chemical germicides.

BACKGROUND: The relative resistance of diverse human bacterial pathogens to commonly used germicidal agents has not been established. METHODS: We measured by titration the survival of thirteen different bacteria after exposure to glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate, sodium hypochlorite, or phenol. RESULTS: Our comparative experiments allowed classification of the organisms' survival into four groups: (a) Pseudomonas aeruginosa and Staphylococcus aureus showed the most resistance, (b) Clostridium perfringens, Salmonella typhimurium, Staphylococcus epidermidis, and Escherichia coli O157:H7 showed intermediate resistance, (c) Listeria monocytogenes, Shigella sonnei, and Vibrio parahaemolyticus survived some treatments with chemical agents only in the presence of protecting protein (serum albumin), and (d) Vibrio cholerae, Vibrio vulnificus, Bacillus cereus, and Yersinia enterocolitica did not survive any of the treatments applied. CONCLUSION: We found species that more frequently survived exposure to germicidal agents were also those most commonly reported in association with hospital infections. Our findings suggest that resistance to disinfectants may be more important than pathogenicity in determining the relative prominence of an organism as an agent responsible for nosocomial infections.

Cleavage of 16S rRNA within the ribosome by mRNA modified in the A-site codon with phenanthroline-Cu(II).

Cleavage of 16S rRNA was obtained through mRNA modified at position +5 with the chemical cleavage agent 1,10-o-phenanthroline. In the presence of Cu2+, and after addition of reducing agent to the modified mRNA-70S complex, cleavage of proximal nucleotides within the 16S rRNA occurred. Primer extension analysis of 16S rRNA fragments revealed that nucleotides 528-532, 1196, and 1396-1397 were cleaved. Nucleotides 1053-1055 were also cleaved but did not show the same level of specificity as the former. These results provide evidence that at some point in the translation process these regions are all within 15 A of position +5, the A-site codon, on the mRNA.

The polymerase chain reaction: applications for the detection of foodborne pathogens.

Faster methods for the detection of foodborne microbial pathogens are needed. The polymerase chain reaction (PCR) can amplify specific segments of DNA and is used to detect and identify bacterial genes responsible for causing diseases in humans. The major features and requirements for the PCR are described along with a number of important variations. A considerable number of PCR-based assays have been developed, but they have been applied most often to clinical and environmental samples and more rarely for the detection of foodborne microorganisms. Much of the difficulty in implementing PCR for the analysis of food samples lies in the problems encountered during the preparation of template DNAs from food matrices; a variety of approaches and considerations are examined. PCR methods developed for the detection and identification of particular bacteria, viruses, and parasites found in foods are described and discussed, and the major features of these reactions are summarized.