Cefixime–tellurite rhamnose MacConkey agar for isolation of Vero cytotoxin-producing Escherichia coli serogroup O26 from Scottish cattle and sheep faeces

Aims:  To compare rhamnose MacConkey agar supplemented with cefixime and tellurite (CT-RMac) and tryptone bile X-glucuronide (TBX) agars as isolation media for Vero cytotoxin-producing Escherichia coli (VTEC) serogroup O26 from animal faeces.
Methods and Results:  Nine VTEC O26 were isolated from sheep faeces; out of which six were isolated only on CT-RMac and one was isolated only on TBX. One hundred and twelve VTEC O26 were isolated from calf faeces; out of which 97% were from CT-RMac and 52% were from TBX. In a study of E. coli O26 strains, 84% of VT-positive O26 did not ferment rhamnose when compared with 16% of VT-negative O26. VT-positive (19%) and VT-negative (39%) E. coli O26 strains did not grow on CT-RMac agar.
Conclusions:  It is important to consider that VTEC O26 strains either may ferment rhamnose or may be sensitive to the CT supplement of CT-RMac agar.
Significance and Impact of the Study:  This work compares CT-RMac and TBX agars as isolation medium for VTEC O26 from Scottish animal faeces and highlights that VTEC O26 may be missed if only CT-RMac agar is used.     

Virulence factors of verocytotoxin-producing Escherichia coli isolated from raw meats.

PCR for verocytotoxin-producing Escherichia coli (VTEC) was positive in 4.6% of 2,440 raw meat samples; only beef, sheep, and venison samples were positive. None of the isolated VTEC strains belonged to serogroup O157. Additional virulence factors were detected in only a minority of strains, suggesting that most of these meat VTEC isolates are not pathogenic.     

Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Comparison of Media for the Isolation of Haemophilus species from Cases of Seasonal Conjunctivitis Associated with Severe Endemic Trachoma

Media for isolation of Haemophilus sp. from the conjuctiva were compared in an oasis in southern Tunisia where severe trachoma and seasonal epidemic purulent conjunctivitis are common. Of 89 children tested, IsoVitaleX-supplemented chocolate agar yielded Haemophilus in 87%, plain chocolate agar in 75%, sheep blood agar with a stab of Staphylococcus epidermidis in 74%, and Fildes medium in 58%. Since other microbial pathogens are easily identified in the modified blood agar, it was the most useful single medium.     

Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli O157: H 7

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.     

Use of prawn blood agar hemolysis to screen for bacteria pathogenic to cultured tiger prawns Penaeus monodon

A newly developed prawn blood agar consisting of 1 ml of tiger prawn hemolymph in medium containing 200 ppm Rose Bengal was used to determine the hemolytic activity of 35 isolates of bacteria obtained from cultured tiger prawns Penaeus monodon and their rearing water. For comparison, the hemolytic activity of these isolates was also determined in sheep blood agar. Nine isolates (25.7% of total) showed different hemolytic reactions on prawn blood agar and sheep blood agar. From the 35 isolates, 8 with various hemolytic characteristics were selected and the relationship between the type of hemolytic activity and pathogenicity was determined and compared. Four isolates that showed hemolytic activity in prawn blood agar caused high mortality to cultured tiger prawns. By contrast, a significantly lower mortality rate was observed for tiger prawns injected with 4 isolates that did not exhibit hemolytic activity on prawn blood agar. Results further showed that mortality did not correlate with hemolytic activity determined using sheep blood agar. Prawn blood agar containing P. monodon hemocytes was faster and more accurate for determining prawn hemolytic activity of bacterial isolates.     

Verotoxin-producing Escherichia coli in sheep grazing an irrigated pasture or arid rangeland forages

Worldwide, verotoxin-producing Escherichia coli (VTEC) have been recognized as the cause of many sporadic cases or major outbreaks of human illnesses involving consumption of contaminated meat, especially beef. Although sheep products have not been linked to reported human illnesses, their role as a food safety risk factor should not be ignored. The objective of this study was to assess VTEC prevalence in two groups of ewes (20 each) grazing an irrigated pasture or arid range in a western United States environment (Nevada) over 1 year (summer of 1999 to summer of 2000). A random sample (n = 504) of potential VTEC isolates were tested for verotoxicity and were screened for the presence (polymerase chain reaction [PCR]) and expression (VTEC-reversed passive latex agglutination assay) of the toxin genes (i.e., VT1 and VT2). Forty-one VTEC isolates (16 having only the VT1 gene and 25 having both VT1 And VT2 genes) were detected in both groups of ewes. Except for seven isolates, the genotype and phenotype data matched. All the isolates (nonmotile [H-]) were non-O157:H7 VTEC (i.e., O91:H- [n = 25], O128:H- [n = 9], and untypeable ones [n = 7]). More infected ewes (nine versus three) and different VTEC strains were found in the irrigated pasture than in the arid range. Because our ewes were shedding two VTEC serotypes known to cause human illnesses, it is beneficial to identify VTEC-positive sheep before slaughter as an initial control point before entering the food chain.     

Subtilase cytotoxin-coding genes in verotoxin-producing Escherichia coli strains from sheep and goats differ from those from cattle

Subtilase cytotoxin (SubAB) from verotoxin (VT)-producing Escherichia coli (VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negative E. coli strains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes (subA and subB). In this study, we investigated the presence of subAB genes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessed subAB genes. The presence of subAB in a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants of subAB. We therefore sequenced the subA gene in 12 strains and showed that the subA gene in most of the subAB-positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while the subA gene in most of the subAB-positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the terms subAB1 to describe the SubAB-coding genes resembling that in the 98NK2 strain and subAB2 to describe those resembling that in the ED 591 strain.     

Haemolysins of Salmonella, their role in pathogenesis and subtyping of Salmonella serovars

Haemolysin patterns of 175 strains of different Salmonella enterica subspecies enterica serovars isolated from different animal sources and places were determined using 11 different blood agar media made with either non-washed horse/sheep erythrocytes or with washed erythrocytes of cattle, sheep, horse, goat, rabbit, guinea pig, and human A, O and B blood groups. Study on 47 strains belonging to 10 serovars of Salmonella from buffalo meat (buffen), 42 strains of 11 serovars from goat meat (chevon): 16 strains of Salmonella enterica serovar Paratyphi B and 25 of S. enterica serovar Paratyphi B var Java from fish, meat, meat products and clinical cases; 45 isolates of S. Abortusequi from aborted mares (18), fetal contents (21), aborted donkey mares (2) and 4 reference strains, revealed that all host restricted Salmonella namely, S. enterica serovar Gallinarum, S. enterica serovar Anatum, S. enterica serovar Abortusequi and S. enterica serovar Paratyphi B could be divided into different haemolysin types based on their inability to produce haemolysis on one or more types of blood agar, while strains of all zoonotic Salmonella serovars induced haemolysis on all the 9 types of blood agar made of washed erythrocytes. None of 175 Salmonella could produce hemolytic colonies on blood agar made of non-washed horse/ sheep erythrocytes. Haemolysin type I (lysing all types of washed erythrocytes) was the commonest one among all serovars except S. Abortusequi, none of which lysed horse erythrocytes. Salmonella enterica serovar Abortusequi having hemolytic activity against sheep erythrocytes were more invasive but had lesser ability to survive in sheep mononuclear cells than non-hemolytic strains. Multiplicity of haemolysins appeared significant epidemiological tool.     

Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.     

Some Microbiological Aspects of Staphylococcal Mastitis

(1) Mannitol fermentation is a reasonably reliable method for the detection of coagulase positive staphylococci in milk. This reliability can be improved if mannitol fermentation is carried out under anaerobic conditions.(2) Among hemolytic strains of staphylococci isolated from milk, beta hemolytic staphylococci predominate. Bovine and sheep blood agar plates give similar hemolytic patterns, but the hemolysis is more pronounced on sheep blood agar.(3) Gelatin liquefaction cannot be relied upon for the selection of coagulase positive staphylococci in milk.(4) Urease production is a feature of the majority of coagulase positive staphylococci isolated from milk.(5) Tellurite Glycine agar medium is not satisfactory for the selection of coagulase positive staphylococci in milk.     

Hair Sheep Blood,Citrated or Defibrinated,Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests

Background

Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis [1], [2]. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.

Methods and Findings

Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.

Conclusions

The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.     

Use of Sodium Polyanethol Sulfonate in the Preparation of 5% Sheep Blood Agar Plates

An alternative method to defibrinating sheep blood for use in bacteriological media is described. The new procedure incorporates sodium polyanethol sulfonate in a concentration of 0.05% (vol/vol). In testing 117 bacterial and fungal isolates, no significant differences were found with respect to adequate growth, pigment production, hemolytic reactions, and other physical attributes. Further tests demonstrate that the sodium polyanethol sulfonate in sheep blood agar plates does not cause any aberrations in zone sizes around disks used in antibiotic susceptibility tests. Consequently, the method represents a suitable alternative to the use of defibrinated sheep blood in the preparation of bacteriological media.     

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.     

Application of a DNA hybridization-hydrophobic-grid membrane filter method for detection and isolation of verotoxigenic escherichia coli.

Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.     

Comparison of PCR, culture and microscopy of blood smears for the diagnosis of anthrax in sheep and cattle

AIMS: To compare microscopy, culture and PCR for the diagnosis of anthrax in blood samples from sheep and cattle. METHODS AND RESULTS: Blood samples were stored at room temperature and at 37 degrees C after receipt, over a period of 15-17 days. Aliquots were plated onto blood agar and blood smears were prepared. Following microscopic examination, DNA was extracted from blood smears and subjected to a multiplex PCR assay targeting the Ba813, cap and lef markers. CONCLUSIONS: PCR provided the most reliable means for the detection of Bacillus anthracis in deteriorating blood samples (15-17 days) and was also successful in diagnosing anthrax in blood smears that had been stored for 6 years and a blood sample which had been stored for 18 months at -20 degrees C. While less successful than PCR, culture for B. anthracis on 7% sheep blood agar was typically more reliable (2-17 days) than the examination of blood smears (2-6 days) for encapsulated bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrated the superiority of PCR for the diagnosis of anthrax from blood smear scrapings, particularly when microscopy is unreliable.     

The type strain(s) of Listeria monocytogenes: a source of continuing difficulties

The type strain of Listeria monocytogenes differs from wild-type L. monocytogenes strains in more characteristics than just the previously reported deficiency in hemolytic activity and virulence in the murine infection model. The type strain from the American Type Culture Collection (strain ATCC 15313) produces lecithinase, is hemolytic on rabbit (but not sheep) blood agar, lacks motility, and shows limited cytopathogenic effects on Caco-2 monolayers, whereas the type strain from the Special Listeria Culture Collection (strain SLCC 53) is unable to produce lecithinase, is nonhemolytic on rabbit or sheep blood agar, is motile, and shows no cytopathogenic effects on Caco-2 monolayers.     

Introduction to the food safety concerns of verotoxin-producing Escherichia coli

Verotoxin-producing Escherichia coli (VTEC) have emerged in the past two decades as food-borne pathogens that can cause major outbreaks of human illnesses worldwide. The number of outbreaks has increased in recent years due to changes in food production and processing systems, eating habits, microbial adaptation, and methods of VTEC transmission. The human illnesses range from mild diarrhea to hemolytic uremic syndrome (HUS) that can lead to death. The VTEC outbreaks have been attributed to O157:H7 and non-O157:H7 serotypes of E. coli. These E. coli serotypes include motile (e.g., O26:H11 and O104:H21) and nonmotile (e.g., O111:H-, O145:H-, and O157:H-) strains. In the United States, E. coli O157:H7 has been the major cause of VTEC outbreaks. Worldwide, however, non-O157:H7 VTEC (e.g., members of the O26, O103, O111, O118, O145, and O166 serogroups) have caused approximately 30% of the HUS cases in the past decade. Because large numbers of the VTEC outbreaks have been attributed to consumption of ruminant products (e.g., ground beef), cattle and sheep are considered reservoirs of these food-borne pathogens. Because of the food safety concern of VTEC, a global perspective on this problem is addressed (Exp Biol Med Vol. 228, No. 4). The first objective was to evaluate the known non-O157:H7 VTEC strains and the limitations associated with their detection and characterization. The second objective was to identify the VTEC serotypes associated with outbreaks of human illnesses and to provide critical evaluation of their virulence. The third objective was to determine the rumen effect on survival of E. coli O157:H7 as a VTEC model. The fourth objective was to explore the role of intimins in promoting attaching and effacing lesions in humans. Finally, the ability of VTEC to cause persistent infections in cattle was evaluated.     

Haemolytic reaction of Listeria monocytogenes on bilayer Columbia Agar plates with defibrinated guinea-pig blood

Haemolysis on blood agar is an important test for the differentation of pathogenic from non-pathogenic Listeria species. The food-borne pathogen Listeria monocytogenes can only be differentiated from harmless L. innocua by its weak β-haemolysis on blood agar. This study demonstrates that L. monocytogenes , which is weakly haemolytic on sheep or horse blood agar forms clear zones of haemolysis on guinea-pig blood agar, which renders haemolysis enhancing methods, such as the CAMP test unnecessary.