Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.     

Sequence Diversity of Bacillus thuringiensis Flagellin (H Antigen) Protein at the Intra-H Serotype Level

In Bacillus thuringiensis, the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes.     

Virulence Patterns in a Murine Sepsis Model of ST131 Escherichia coli Clinical Isolates Belonging to Serotypes O25b:H4 and O16:H5 Are Associated to Specific Virotypes

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.     

Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan

Of 809 soil samples collected from the seven islands of the Ryukyus, Japan, 107 samples (13.2%) contained Bacillus thuringiensis. The frequency of B. thuringiensis among the B. cereus group was 1.1% (235/21842) on the average. The B. thuringiensis soil populations of the Ryukyus consisted of more than 22 H serogroups. The predominant H serotype was the H5ac/21 (serovar canadensis/colmeri), followed by the H3ad (serovar sumiyoshiensis) and H16 (serovar indiana). Geographically, most widely distributed H serogroups were the H16 and H10ac (serovar londrina); the former was recovered from five islands and the latter from three islands. Parasporal inclusions of the isolates were morphologically heterogeneous, roughly grouped into four categories: bipyramidal/cuboidal, spherical/ovoid, irregularly-pointed, and irregular-shaped. About 53% of the isolates formed spherical to ovoid parasporal inclusions. None of the isolates exhibited larvicidal activity against the silkworm, Bombyx mori. Only four isolates belonging to four different serotypes killed larvae of the mosquito, Aedes aegypti. These mosquito-specific isolates all produced spherical parasporal inclusions.     

Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries

Mulberry leaves were examined for the occurrence of Bacillus thuringiensis. This organism was recovered from both abaxial and adaxial surfaces: a total of 186 B. thuringiensis colonies were isolated from 24 (96·0%) out of 25 mulberry trees, and from 112 (11·2%) out of 1004 leaves from 25 trees. The frequency of B. thuringiensis colonies was 3·2% among 5900 colonies belonging to the Bacillus cereus/B. thuringiensis group. Single colonies were associated with 75·9% of the B. thuringiensis -positive leaves and 2–16 colonies were occasionally found on a single phylloplane. Flagellar (H) serotypying of the isolates revealed that, among the 19 H serotypes (serovars) detected, the H serotype 13 (serovar pakistani ) was the predominant, followed by the H serotypes 3abc ( kurstaki ), 6ac ( oyamensis ), 16 ( indiana ), 24 ( neoleonesis ), 4ac ( kenyae ), 7 ( aizawai ) and 10 ( darmstadiensis ). Larvicidal activity, against the silkworm ( Bombyx mori ) and/or the mosquito ( Aedes aegypti ), was exhibited by 18 isolates (9·7%) belonging to H serovars kurstaki, kenyae, canadensis and aizawai , and an unidentified H serogroup.     

Serotypes, virulence genes, and PFGE patterns of enteropathogenic Escherichia coli isolated from Cuban pigs with diarrhea.

Thirty-six enteropathogenic Escherichia coli strains isolated from Cuban pigs with diarrhea were serotyped and screened by PCR for the presence of virulence genes. The 36 isolates belonged to 11 O serogroups and 14 O:H serotypes, with 53% of the isolates belonging to only two serotypes: O141:H- (13 isolates) and O157:H19 (6 isolates). Genes coding for STb, STa, VT2e, and LT toxins were identified in 69, 61, 53, and 6% of the isolates, respectively. The most prevalent fimbrial adhesin was F18, detected in 22 (61%) isolates. The gene encoding F6 (P987) colonization factor was identified in three (8%) isolates. None of the 36 isolates assayed contained genes encoding F4 (K88), F5 (K99), or F41. The seropathotype O141:H-:STa/STb/VT2e/F18 (13 isolates) was the most frequently detected, followed by O157:H19:VT2e/F18 (5 isolates). A genetic diversity study, carried out by pulsed-field gel electrophoresis (PFGE) of 24 representative isolates, revealed 21 distinct restriction patterns clustered in 18 groups (I-XVIII). Isolates of the same serotype were placed together in a dendrogram, but isolates of serotype O157:H19 showed a high degree of polymorphism. The results of this study demonstrate the presence in Cuba of different clusters among one of the most prevalent serotypes isolated from pigs with diarrhea. Further experiments are needed to determine whether some of these clusters have appeared recently; if so, their evolution, as well as their possible association with pathogenicity in farms should be studied.     

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Comparative study of the frequency and flagellar serotype flora of Bacillus thuringiensis in soils and silkworm-breeding environments

Two environments, soils and silkworm ( Bombyx mort )-breeding farms, in Kumamoto Prefecture, Japan, were explored for the comparative analysis of the frequency and the flagellar (H) serotype flora of Bacillus thuringiensis. This organism was found in 39.3% of the sericulture farms and 18.5% of the silkworm litter samples. The frequency of B. thuringiensis was 4.5% among the populations of the Bacillus cereus/B. thuringiensis group in silkworm-breeding insectaries. The predominant H serotype detected in silkworm litters was 4ac (35.6%), followed by serotypes 3abc (20.0%), 8ab (13.3%), 4ab (13.3%) and 7 (2.2%). The majority (87.1%) of sericulture-derived isolates exhibited larvicidal activity against the silkworm and/or the mosquito, Aedes aegypti. Of soil samples examined, 8.1% contained B. thuringiensis. The frequency of B. thuringiensis colonies was 0.6% among soil populations of the B. cereus/B. thuringiensis group. Of 22 soil isolates, 14 were assigned to five H serotypes: 10, 17, 18, 21 and 29. Most soil isolates showed no insecticidal activity against B. mori and A. aegypti , but two, belonging to H serotype 10 and an undescribed serotype, exhibited moderate larvicidal activity against A. aegypti.     

Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens.

A total of 525 Bacillus thuringiensis environmental isolates, belonging to the five flagellar (H) serovars (alesti, sotto, kenyae, aizawai, and morrisoni), were serotyped by extracellular heat-stable somatic antigens (HSSAs). The isolates belonging to a given H serovar were assigned to a single HSSA serogroup at a high frequency, 87-100%. This indicates that the extent of HSSA variation within a single H serovar is small in the field populations of these B. thuringiensis serovars.     

Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).

The lipopolysaccharide (LPS) of Klebsiella serotype O2 is antigenically heterogeneous; some strains express multiple antigenic factors. To study this heterogeneity, we determined the structure of the O-antigen polysaccharides in isolates belonging to serotypes O2(2a), O2(2a,2b), and O2(2a,2c), by using composition analysis, methylation analysis, and both 1H and 13C nuclear magnetic resonance spectroscopy. The repeating unit structure of the 2a polysaccharide was identified as the disaccharide [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] and was identical to D-galactan I, one of two O polysaccharides present in the LPS of Klebsiella pneumoniae serotype O1 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). LPS from serotype O2(2a,2b) also contained D-galactan I as the only O polysaccharide, suggesting that the 2b antigen is not an O antigen. The LPS of serotype O2(2a,2c) contained a mixture of two structurally distinct O polysaccharides and provides a second example of this phenomenon in Klebsiella spp. One polymer was identical to D-galactan I, and the other polysaccharide, the 2c antigen, was a polymer with a disaccharide repeating unit structure, [----3)-beta-D-GlcpNAc-(1----5)-beta-D-Galf-(1----]. The 2c structure does not resemble previously reported O polysaccharides from Klebsiella spp. Periodate oxidation confirmed that D-galactan I and the 2c polysaccharide are distinct glycans, rather than representing domains within a single polysaccharide chain. Monoclonal antibodies against the 2c antigen indicated that only LPS molecules with the longest O-polysaccharide chains contained the 2c epitope.     

Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar.

Clamped homogeneous electric field (CHEF) electrophoresis was optimized for genomic analyses of Listeria monocytogenes. Various human, animal, food, and environmental isolates, as well as strains representing other Listeria species, were separately digested with rarely cutting endonucleases. Of 176 L. monocytogenes strains analyzed, the enzymes AscI and ApaI established 63 and 72 unique restriction endonuclease digestion profiles (REDP), respectively. The 22 non-L. monocytogenes strains exhibited 18 AscI and 19 ApaI unique REDP. Statistical analyses of REDP information using the Dice coincidence index and principal component analysis revealed two distinct genomic divisions of L. monocytogenes that also correlated with the flagellar (H) antigen type: division I contained serovar 1/2a, 1/2c, 3a, and 3c stains and division II contained serovar 1/2b, 3b, 4b, 4d, and 4e strains. Division I isolates digested with ApaI were further grouped into cluster IA (serovar 1/2c and 3c) and cluster IB (serovar 1/2a and 3a) strains. Likewise, division II isolates digested with ApaI were further grouped into cluster IIA (serovar 1/2b and 3b) and cluster IIB (serovar 4b, 4d, and 4e) strains. These data indicate that genotypic data generated by CHEF can be directly related to phenotypic data generated by serotyping for establishing the overall relatedness of isolates. Moreover, these data further substantiate that CHEF analysis is a reproducible and highly discriminating method for characterizing L. monocytogenes strains at the molecular level.     

Bacillus thuringiensis: A Common Member of Microflora in Activated Sludges of a Sewage Treatment Plant

Bacillus thuringiensis was recovered at a high frequency from activated-sludge system environments in an urban sewage-digestive plant. All of the test materials, sampled at several digesting steps, contained the organism. Of 515 colonies belonging to the B. cereus/B. thuringiensis group, 45 (8.7%) were assigned to B. thuringiensis. The highest density of this bacterium was 1.6 × 10³ cfu/ml in a scum sample of the first aeration basin. Among the 45 isolates, 7 were assigned to the known H serovars. Two isolates of the serovar kenyae isolates exhibited Lepidoptera-specific toxicity. Diptera-specific toxicity was shown by an isolate of serovar israelensis and a serologically undefined isolate. Lectin activity was associated with 12 isolates. Received: 10 October 2000 / Accepted: 17 November 2001     

Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized. Received: 3 December 1999 / Accepted: 5 January 2000     

Bacillus thuringiensis subspecies huazhongensis, serotype H40, isolated from soils in the People's Republic of China

DAI JINGYUAN, YU LING, WANG BO, LUO XIXIA, YU ZINIU AND M.-M. LECADET. 1996. Two isolates (YBt-981 and Ybt-978) of Bacillus thuringiensis were isolated from soil samples from Shanxi and Neimeng provinces in China. The isolates produced small and irregular parasporal inclusions that were not toxic to larvae of Culex fatigans, Culex pipiens, Anopheles sinensis, Aedes aegypti, Spodoptera littoralis, Spodoptera exigua, Plutella xylostella and Bombyx mori . H-antigens from isolates of YBt-981 and YBt-978 differed from those of the known B. thuringiensis H1 to H39 serotypes. YBt-981 and YBt-978 had the same H-antigens and the same biochemical characters. The two isolates were identified as a new serotype designated H40. The name of B. thuringiensis serovar huazhongensis for this new subspecies represented by YBt-981 and YBt-978 is proposed.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders, Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage. Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.     

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae

Abstract Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognize serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG_2a, IgG_2b, and IgG₃ were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.     

Distribution of the four flagellar (H) antigenic subserotypes of Bacillus thuringiensis H serotype 3 in Japan

A total of 302 strains of Bacillus thuringiensis flagellar (H) serotype 3, collected from sericultural environments and soils of the three main islands (Honshu, Shikoku and Kyushu) of Japan, were examined for their H antigenic subserotypes. Of these strains, 259 (85.8%) were identified as the H subserotype 3a : 3c (subsp. alesti ), 16 (5.3%) were referable to the subserotype 3a : 3b : 3c (subsp. kurstaki ), 26 (8.6%) belonged to the subserotype 3a : 3d : 3e (subsp. fukuokaensis ) and one strain (0.3%) was the subserotype 3a : 3d (subsp. sumiyoshiensis ). The subserotypes 3a : 3c and 3a : 3b : 3c were present in sericultural environments only; the former was distributed in Honshu and Shikoku Islands but not in Kyushu Island, and the latter was distributed in Honshu and Kyushu Islands but not in Shikoku Island. The subserotype 3a : 3d : 3e, mainly found in soils but occasionally in sericultural environments, was distributed in the three main islands. It also appeared that the subserotype 3a : 3d : 3e, a recently described subserotype, was distributed in Japan more widely than the globally disseminated subserotype 3a : 3b : 3c. The results clearly showed geographical and ecological differences in the distribution of subserotypes.     

Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples.     

Population genetics and antibiotic susceptibility of invasive Haemophilus influenzae in Manitoba, Canada, from 2000 to 2006

One hundred and twenty-two isolates of Haemophilus influenzae causing invasive disease were collected in Manitoba, Canada, from 2000 to 2006 and examined for serotype, biotype, sequence type (ST) by multilocus sequence typing and antibiotic susceptibility. Nonserotypeable (NST) isolates accounted for over half of the isolates collected (69 isolates, 56.6%). There were 36 serotype a, five serotype b, two serotype c, one serotype d, four serotype e and five serotype f isolates collected. The 69 NST isolates were found to be very diverse, with isolates representing six biotypes and 45 STs. The serotypeable isolates were more clonal, with each of the serotypes showing little diversity in their biotypes and STs. Of the 122 isolates, 17% were resistant to ampicillin due to beta-lactamase production, 10.7% were resistant to trimethoprim-sulfamethoxazole, 1.6% were resistant to clarithromycin, 2.5% were resistant to amoxicillin-clavulanic acid and none was resistant to ciprofloxacin or moxifloxacin. Antibiotic resistance was more common in the NST strains, with 37.7% showing resistance to at least one antibiotic compared to 15% in the serotypeable strains. The results of this study suggest a shift in the epidemiology of invasive H. influenzae infections in the post-Hib vaccine era, and surveillance should include all serotypeable and NST isolates.     

Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina.

A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type e1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type q/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC.