Comparison of the Recovery of Escherichia coli from Frozen Foods and Nutmeats by Confirmatory Incubation in EC Medium at 44.5 and 45.5 C

The productivity of confirmatory EC broth for the isolation of fecal Escherichia coli was determined at 44.5 and 45.5 C. A variety of frozen pre-cooked foods and an assortment of nutmeats were examined after primary incubation in Lauryl Sulfate Tryptose (LST) broth. In 85.3% of the cases, the parallel tubes of EC broth incubated for 24 hr at 44.5 and 45.5 C gave rise to identical E. coli responses of positive, false positive, and negative. The remaining 14.7% of the reactions represent the qualitative difference between the two temperatures. The EC test at 45.5 C was more specific for E. coli, since two- to threefold fewer false positives were produced at this temperature than at 44.5 C. However, fecal E. coli recoveries were slightly higher (4%) at the lower temperature. Incubating the EC tubes from the interval of 24 to 48 hr gave rise to an additional 4.3% of E. coli recovery, but this was accompanied by an excessive production of false positives (75.9%), representing a 3.5-fold decrease in specificity. It is recommended that, in the confirmatory use of EC broth in the examination of frozen foods and nutmeats for the recovery of fecal E. coli, the test be made at 45.5 C in a water bath and limited to 24 hr of incubation only, to insure optimal specificity. During the study, a “fixed” productivity ratio was noted; E. coli⁺/LST⁺ equaled approximately one-fourth or 25%. The significance of this ratio is discussed.     

Characterization of Staphylococci Isolated from Raw Milk

To evaluate the pathogenicity of staphylococci from bovine raw milk, the general characteristics of 775 strains isolated from 798 samples of milk were studied. The coagulase test was performed by use of rabbit plasma. Chromogenesis, mannitol fermentation, and gelatin liquefaction were investigated on Chapman's Medium 110, after 48 hr of incubation. Production of β-hemolysin, which has been considered indicative of pathogenic staphylococci of animal origin, was determined by streaking different strains on sheep blood-agar plates in the presence of a strain of Lancefield group B streptococci. Plates were incubated at 37 C for 24 hr, and strong hemolysis was produced in the zone of interaction of β-hemolysin and some substance liberated by streptococcus (CAMP test). Of 404 strains found to be coagulase-positive, 95.8% exhibited a deep-orange pigment, 76.5% produced β-hemolysin, 91.8% fermented mannitol, and 75% liquefield gelatin. Of 371 strains which gave a negative coagulase test, about 16% fermented mannitol and liquefied gelatin; none of these strains produced β-hemolysin. When results are grouped according to pigmentation and coagulase production, β-hemolysin seems to be developed by pathogenic strains of Staphylococcus aureus only. If suitability of these tests for investigation of pathogenicity is compared, production of β-hemolysin appears to be the most useful one, since no “false positive” results were found. The use of the CAMP test as a simple and rapid technique to determine production of β-hemolysin by pathogenic strains of animal staphylococci during routine bacteriological work is suggested.     

Genetic Characterization of Escherichia coli Populations from Host Sources of Fecal Pollution by Using DNA Fingerprinting

Escherichia coli isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting. The genetic relationship of strains within each host group was assessed as was the relationship of strains among different host groups. Multiple isolates from a single host animal (gull, human, or dog) were found to be identical; however, in some of the animals, additional strains occurred at a lower frequency. REP PCR fingerprint patterns of isolates from sewage (n = 180), gulls (n = 133), and dairy cattle (n = 121) were diverse; within a host group, pairwise comparison similarity indices ranged from 98% to as low as 15%. A composite dendrogram of E. coli fingerprint patterns did not cluster the isolates into distinct host groups but rather produced numerous subclusters (approximately >80% similarity scores calculated with the cosine coefficient) that were nearly exclusive for a host group. Approximately 65% of the isolates analyzed were arranged into host-specific groups. Comparable results were obtained by using enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis (PFGE), where PFGE gave a higher differentiation of closely related strains than both PCR techniques. These results demonstrate that environmental studies with genetic comparisons to detect sources of E. coli contamination will require extensive isolation of strains to encompass E. coli strain diversity found in host sources of contamination. These findings will assist in the development of approaches to determine sources of fecal pollution, an effort important for protecting water resources and public health.     

Antibiotic resistance and detection of β-lactamase in bacterial strains of Staphylococci and Escherichia coli isolated from foodstuffs

Seventy strains of Staphylococcus spp. and Escherichia coli (35 each) were isolated from various foodstuffs and identified on the basis of cultural, morphological and biochemical characteristics and were further tested for their antibiotic susceptibility with commonly used antibiotics/drugs. 69.2% of the strains of Staphylococci were resistant to co-trimazine and 34.6% were resistant to penicillin-G. 19.2% of the staphylococcal isolates exhibited resistance to cloxacillin, nalidixic acid, methicillin and tetracycline whereas 15.3% of the staphylococcal isolates were resistant to amoxycillin and nitrofurantoin. The isolated E. coli strains exhibited sharp peaks of resistance to antimicrobial agents such as tetracycline (72%), doxycycline (60%) and nalidixic acid (48%). Forty-four percent of the E. coli strains were resistant to nitrofurantoin and penicillin-G respectively. Among the 13 antibiotics/drugs tested for resistance, six different resistance patterns were observed in staphylococcal isolates and seven different resistance patterns were observed in the E. coli isolates from various foodstuffs. Bacterial strains exhibiting MIC values 100 g/ml for ampicillin and cloxacillin were screened for -lactamase activity and out of 10 staphylococcal isolates, seven were found to be positive for -lactamase, whereas out of 13 E. coli isolates tested for -lactamase production, only three were found to be positive.     

Prevalence, Antibiotic Susceptibility, and Diversity of Escherichia coli O157:H7 Isolates from a Longitudinal Study of Beef Cattle Feedlots

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.     

The Aerogenic Response of Escherichia coli and Strains of Aerobacter in EC Broth and Selected Sugar Broths at Elevated Temperatures

Gas formation by 116 strains of Escherichia coli and 104 strains of Aerobacter was determined in a specially constructed and accurately controlled water bath employing EC, lactose, maltose, sucrose, glucose, levulose, and galactose broths at temperatures ranging from 44.5 to 46.5 C.

Greatest gas activity occurred in EC broth. In the range 44.9 to 45.5 C over 92% of the E. coli cultures formed gas, but the Aerobacter strains dropped from 68 to 2%. A natural point of separation of the two groups occurred at 45.5 C.

Inhibition of the gas-forming mechanism rather than death is the universal response of the Escherichia organisms to these temperatures. The inhibition increases with rising temperatures and is readily reversible. At 46.5 C, 64.5% of all the Escherichia cultures were inhibited and 69.1% of all the cultures were actually viable.

In EC broth it was found that as a group atypical E. coli (-+--) were the most resistant gas-positive types. Least resistant in EC broth was a group of known typical fecal isolates of E. coli (++--). Of intermediate resistance between the two groups was the large body of typical E. coli (++--) organisms.

Certain individual strains of E. coli excelled in the production of gas in the variety of sugar broths tested at elevated temperatures. The Aerobacter strains did not exhibit this property.

Finally it is suggested that elevated temperature incubation studies of this type be conducted in critically controlled water baths with an ascertained accuracy in the vicinity of 45.5 ± 0.1 C under full load.

     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Conjugal transfer of natural plasmids between Escherichia coli strains in sterile environmental water

Seven antibiotic-multiresistant Escherichia coli strains, possessing three or four plasmids, capable of transferring their resistance marker at a high frequency, were selected among a total of 300 antibiotic-resistant E. coli strains isolated from natural water—raw and treated wastewater, and brackish water (collected 1 km downstream). These strains were mated with E. coli K-12 C600 nal^r, both in sterilized natural water and LB medium at 25°C. Conjugation did occur in all the systems tested, although fewer transconjugants were recovered from raw and treated wasterwater experiments. In contrast, in brackish and seawater, the transfer frequency did not significantly decrease in spite of salt contents. In 100% of the cases, transfer of the high-molecular-weight plasmids (20 kb) was observed, but the small plasmids (2.6–7.5 kb) were only cotransferred in raw or treated wastewater and in brackish water. Moreover, genotypic variation occurred more frequently in natural water than in LB medium.     

Extended spectrum beta-lactamases in <Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from homemade white cheeses: prevalence and antibiotic susceptibility

The present study included 223 E. coli strains isolated from homemade white cheeses and was undertaken to determine the prevalence of extended spectrum β-lactamases (ESBL)-producing strains and antibiotic susceptibility. ESBL production was detected using three methods: the NCCLS disk diffusion test, the double disk synergy test and the NCCLS confirmatory test. By the disk diffusion test, 48% isolates were determined as potential ESBL producers. ESBL production by the double disk test and the phenotypic confirmatory test was found to be 16.1 and 9.9%, respectively. Susceptibility of all isolates against 16 antimicrobials was tested by the disk diffusion method. All strains were imipenem- and cefepime-susceptible. Susceptibility of E. coli to ceftazidime, ceftriaxon, cefotaxime and aztreonam was found to be 93.7, 96.4, 81.2, 90.6%, respectively. The least effective antibiotics were ampicillin with a rate of 68.6% and cefuroxime with a rate of 69.1%.     

Rapid Recovery of Escherichia coli from Estuarine Water

The efficiencies of two 24-hr elevated-temperature tests to recover Escherichia coli from estaurine water were compared simultaneously with the 72-hr standard methods procedure of the American Public Health Association (APHA). From 1,710 tubes, E. coli was recovered 222 times in lauryl tryptose medium incubated at 44 ± 0.2 C for 24 hr, 261 times in an experimental medium incubated at 44.5 ± 0.2 C for 24 hr, and 257 times by the 72-hr APHA method. The number of false positives enumerated was similar in all three tests. The data indicated that E. coli in raw seawater could be determined in 24 hr without a significant loss of accuracy.     

Distribution and Diversity of Escherichia coli Populations in the South Nation River Drainage Basin,Eastern Ontario,Canada

We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 × 10⁵ CFU 100 ml⁻¹ and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.Escherichia coli is ubiquitously distributed in fecal material from humans and warm-blooded animals (38). The detection of E. coli in water is an implicit indicator of recent fecal contamination and therefore of the risk of cooccurrence of enteric pathogens that can cause illness in susceptible populations (62). Many jurisdictions evaluate and mandate compliance with drinking and recreational water quality standards on the basis of the presence and abundance of E. coli (14, 44). For example, Canadian recreational water quality standards stipulate that E. coli densities in excess of a geometric mean of 200 CFU per 100 ml indicate that the water is unsuitable for swimming and bathing (23).In a background of increasing occurrence of microbial contamination of surface water, a variety of methods for elucidating the sources of fecal contamination have been developed, and these microbial source tracking (MST) methods are recommended components of fecal pollution abatement strategies (16, 57). So-called library-dependent MST methods compare environmental isolates to collections of isolates obtained from likely sources of fecal pollution in the area of investigation. The host source is distinguished on the basis of the similarity of environmental isolates to reference fecal isolates. Comparison can be undertaken on the basis of genomic fingerprinting methods, including repetitive extragenic palindromic PCR (rep-PCR), ribotyping, or pulsed-field gel electrophoresis (PFGE) (13, 17, 31, 54, 57). A variety of studies using these methods have revealed enormous diversity in the fecal and environmental E. coli populations. For example, 461 distinct PFGE genotypes and 175 distinct enterobacterial repetitive intergenic consensus (ERIC)-PCR genotypes were detected in a collection of 555 E. coli strains isolated from river water in Texas (10). As many as 291 and 94 rep-PCR genotypes were distinguished in collections of 643 river isolates and 353 beach water E. coli isolates, respectively (43). Significant diversity was also revealed using multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) on 185 E. coli isolates from freshwater beaches, where an average of 40 alleles per locus were detected (59). Almost 60% of 657 E. coli isolates in a fecal reference collection had unique (i.e., detected in only one individual) fingerprints determined by rep-PCR (32). Extensive diversity of E. coli was also observed in soils in temperate climates, where the growth and persistence of “naturalized” populations without any known fecal input have been found (7, 28, 30). Naturalized populations have been dominated by the B1 phylogroup and may have adapted in ways that enhance their survival in temperate secondary habitats (59). The temporal and spatial diversity of E. coli may not be a significant factor in coarse-source (e.g., human versus animal) classification of E. coli by means of ribotyping procedures (48). Ultimately, the characterization and understanding of the diversity of populations of selected microorganisms in surface watercourses affected by multiple sources of fecal pollution (as in agricultural watershed settings, for example) may be more critical for assessing the specific impacts of contamination-mitigating measures than previously thought. For instance, restricting the access of cattle on pasture to adjacent water by implementing vegetative buffering along watercourses creates habitat for varied wildlife, which then contribute to fecal pollution. In this context, the diversity in populations of indicator bacteria could be useful for better understanding how changes in landscape use influence fecal source inputs.As part of a research program evaluating the impact of agriculture on water quality and the efficacy of better agricultural management practices to mitigate agricultural pollution, we have conducted a multiyear study of the microbiological water quality for a suite of different-sized watersheds in the South Nation River basin in eastern Ontario, Canada (41, 46, 61). Land use in this river basin is mixed, consisting primarily of agricultural activities, light urban development, and interspersed wildlife habitat. Surface water systems in the study region differ widely in their contributing areas and therefore in their discharges (61).In the work undertaken here, we sought to determine the spatial and seasonal variability in the density and the structure of populations of E. coli in surface waters within the South Nation River basin. The specific objectives of the study were (i) to characterize the seasonal distribution and abundance of E. coli in different watershed settings within the river basin, (ii) to evaluate the spatial distribution of E. coli densities and diversity with respect to upstream land use activities, (iii) to use rep-PCR to elucidate the dominant E. coli genotypes and the diversity of E. coli populations and to explore linkages to pathogen presence, season, and environmental and land use variables, and (iv) using rep-PCR, to evaluate the concordance between waterborne isolates and fecal isolates obtained from within the study area. The study is distinguished by an intensive 4-year sampling of numerous (n = 24) sites that differed in their stream order and proximal land use activity; the number of E. coli isolates (≈21,000) included in the analysis; and the use of two distinct rep-PCR fingerprinting methods (ERIC and BOXA1R) to characterize the isolates. Furthermore, we used classification and Regression Tree (CART) analysis to evaluate relationships between the abundance and diversity of E. coli in water samples and environmental and land use variables.     

Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex

Summary Two yeast DNA pools inserted in an hybridEscherichia coli-yeast vector pFL1 were used to transformE. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool — aBamHI yeast DNA digest — a 6.4 kbBamHI fragment was recovered that gave good complementation of theE. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool — a partialSau3A yeast DNA digest — five independent plasmids complementing eitherE. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kbBamHI fragment. One of these plasmids, which complemented the twoURA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the fullURA2 gene. A restriction map of theURA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization withURA2 messenger RNA, allowing us to estimate the limits of the gene.     

Potentially Pathogenic Escherichia coli Can Form a Biofilm under Conditions Relevant to the Food Production Chain

The biofilm-producing abilities of potentially human-pathogenic serotypes of Escherichia coli from the ovine reservoir were studied at different temperatures and on different surfaces. A possible influence of the hydrophobicity of the bacterial cells, as well as the presence of two virulence factors, the Shiga toxin-encoding (Stx) bacteriophage and the eae gene, was also studied. A total of 99 E. coli isolates of serotypes O26:H11, O103:H2, and O103:H25 isolated from sheep feces were included. The results show that isolates of all three E. coli serotypes investigated can produce biofilm on stainless steel, glass, and polystyrene at 12, 20, and 37°C. There was a good general correlation between the results obtained on the different surfaces. E. coli O103:H2 isolates produced much more biofilm than those of the other two serotypes at all three temperatures. In addition, isolates of serotype O26:H11 produced more biofilm than those of O103:H25 at 37°C. The hydrophobicity of the isolates varied between serotypes and was also influenced by temperature. The results strongly indicated that hydrophobicity influenced the attachment of the bacteria rather than their ability to form biofilm once attached. Isolates with the eae gene produced less biofilm at 37°C than isolates without this gene. The presence of a Stx bacteriophage did not influence biofilm production. In conclusion, our results show that potentially human-pathogenic E. coli from the ovine reservoir can form biofilm on various surfaces and at several temperatures relevant for food production and handling.     

反刍动物产志贺毒素大肠杆菌的分离鉴定和致病潜力分析

产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）是重要的食源性病原,而STEC往往以正常菌群的形式存在于牛羊等反刍动物肠道。[目的] 本研究对牛羊粪便样品中的STEC分离和鉴定并对分离株进行致病潜力分析。从江苏、云南和河北等地共分离到羊源STEC菌株11株,牛源STEC菌株1株,另新疆农业大学佟盼盼组馈赠牛源菌株10株。[方法] 通过细菌选择培养及特异性基因stx1和stx2的检测进行分离鉴定;并通过Vero细胞毒性试验、溶血活性试验和毒力因子的检测分析STEC分离株的致病潜力。[结果] 分离到羊源分离株11株,分离率17.5%（11/63）;分离得到牛源分离株1株,分离率0.7%（1/134）;11株羊源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性;11株牛源分离株中有5株对Vero细胞具有强的毒性,3株有溶血活性。11株羊源STEC分离株eae基因携带率为63.6%（7/11）,而11株牛源STEC分离株eae基因携带率仅为9.0%（1/11）。[结论] 结果表明羊源STEC菌株的分离率和致病潜力高于牛源菌株,所以,除牛外,羊作为STEC菌株宿主也应该得到更多的重视。     

The effect of ampicillin on the electrophysical properties of Escherichia coli cells

The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OSs) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10–1000 kHz of the orienting electric field and does not affect the OSs of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 µg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OSs of cells can be used to evaluate their resistance to ampicillin.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 126–131.Original Russian Text Copyright © 2005 by Guliy, Markina, Ignatov, Shchegolev, Zaitseva, Bunin, Ignatov.     

Examination of Feces from Food Handlers for Salmonellae, Shigellae, Enteropathogenic Escherichia coli, and Clostridium perfringens

Duplicate fecal specimens from food handlers were collected in Louisiana. One set of specimens was examined immediately for salmonellae and shigellae by the Central Laboratory of the Louisiana State Board of Health in New Orleans; the other set was shipped to the Food Microbiology Unit at the Robert A. Taft Sanitary Engineering Center in Cincinnati, Ohio, where it was examined for enteropathogenic Escherichia coli (EEC) and Clostridium perfringens. A total of 219 specimens were examined by both laboratories. None yielded salmonellae or shigellae; 171 (78.1%) yielded C. perfringens; 175 (79.9%) yielded E. coli; and 14 (6.4%) yielded EEC. The 14 isolates of EEC were distributed among eight serotypes; one specimen yielded two serotypes. Multiple isolations of C. perfringens strains (two to four) were made from 64 (37.4%) of the specimens, and a total of 244 strains were isolated and studied for identifying characteristics. Of the total, only 87 (35.5%) could be identified serologically by a battery of 67 antisera; only 4 (1.6%) possessed the characteristics of the English “food-poisoning type.” The hemolytic activity on agar containing horse, ox, or sheep blood showed that 140 (57.1%) were “hemolytic,” 81 (33.1%) were “nonhemolytic,” and 23 (9.8%) gave varied results. Only 12 (4.9%) of the strains produced spores that resisted boiling for 30 min or more.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.     

Enterotoxigenic Bacteria in Food and Water from an Ethiopian Community

Food and water samples from an Ethiopian community were screened for the presence of enterotoxin-producing bacteria. Using the Chinese hamster ovary cell assay, 40 of 213 isolates (18.8%) produced heat-labile (LT) enterotoxin. These LT-producing isolates comprised 33 of 177 (18.6%) strains from 24 of 68 food samples (35.3%) and 7 of 36 (19.4%) isolates of 4 of 17 water samples (23.5%). One LT-producing strain each of Salmonella emek and of Shigella dysenteriae was found. Three pseudomonads, all LT producers, produced heat-stable enterotoxin as gauged by the suckling mouse test. Two strains of LT-enterotoxigenic Escherichia coli O68 were found in water samples. No enterotoxigenic E. coli were isolated from food samples, but 13 of the LT-producing strains were Enterobacter, Klebsiella, Serratia, and Proteus species, and 7 food samples yielded more than one species of enterotoxigenic bacterium. Of the enterotoxigenic isolates from food, 15 were oxidase-positive strains of the genera Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. LT-enterotoxigenic Enterobacter, Acinetobacter, Klebsiella, Proteus, Providencia, and Serratia species represented 20 of the food and water isolates. Culture supernatant fluids of representative strains of oxidase-positive and oxidase-negative species giving positive reactions in Chinese hamster ovary cell tests induced fluid accumulation in rabbit ileal loops. Eight of the food samples and two of the water samples contained more than one isolate or species of enterotoxigenic bacterium. The stability of the LT production by oxidase-positive bacteria and non-E. coli strains was assessed by the rabbit skin and adrenal cell tests after 9 months and 1 year of storage, respectively, in Trypticase soy broth with glycerol at −70°C. Only 33% of the oxidase-positive strains were still LT enterotoxigenic. Of the oxidase-negative strains, 50 and 33% were LT producing at 9 months and 1 year, respectively. None of the E. coli isolates, both enterotoxigenic and nonenterotoxigenic, possessed K88, K99, or colonization factor antigen. The survey demonstrates the presence in food and water of enterotoxigenic bacteria of the same species as those isolated from cases of infantile diarrhea in the same community, although a correlation between these sources and infantile diarrhea remains to be established.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Production of Monoclonal Antibody Discriminating Serological Difference in Escherichia coli O9 and O9a Polysaccharides

A monoclonal antibody (mAb) with a unique antigenic specificity against Escherichia coli O9 was produced. The O9a mAb was reactive with a part of the strains in E. coli O9. The O9a mAb did not react with LPS from the E. coli O9 test strain Bi316-42. The distribution of the antigen defined by the O9a mAb in E. coli O9 was consistent with that of E. coli O9a present in E. coli O9 strains. The chemical structure of the repeating unit of the O-specific polysaccharide detected by the mAb was demonstrated to be a mannotetraose by two-dimensional nuclear magnetic resonance spectroscopy. It was confirmed that the mAb recognized E. coli O9a serotype in E. coli O9 serotype strains, suggesting that E. coli O9a serotype might be a dominant strain in E. coli O9.