Detection, quantitation and stability of the beta haemolysin of Aeromonas spp

Amongst 58 isolates of motile aeromonads evaluated for the ability to produce beta haemolysin, haemolytic activity was significantly associated with strains belonging to the Aeromonas hydrophila and A. sobria groups. Of erythrocytes from nine animal species tested, mouse red blood cells provided the best indicator system for detection of beta-haemolysin activity. Furthermore, differences in the stability of the beta haemolysins of selected A. sobria and A. hydrophila isolates at different temperatures, and in the presence of urea or dithiothreitol were observed.     

Enterotoxin production by Staphylococcus aureus isolates from cases of septicaemia and from healthy carriers

In a prospective study, 52 Staphylococcus aureus isolates from individual patients with septicaemia and 27 nasal strains from separate, healthy carriers were compared for production of a range of extracellular proteins and toxins. Whereas there was no difference (p greater than 0.05) between septicaemic and nasal isolates with respect to incidence of alpha, beta, gamma and delta haemolysins, toxic shock syndrome toxin-1 or staphylokinase production, the incidence of enterotoxin A, B, and C production was higher among isolates from septicaemia (p less than 0.01). Of the isolates from septicaemia, 33 (63%) produced enterotoxins A, B, C or D alone or in combination. Only three (11%) of the nasal isolates produced a single enterotoxin, enterotoxin D. Of the isolates from septicaemia, 67% were hospital-acquired and greater than 25% of these were endemic, methicillin-resistant (MRSA) strains. All MRSA strains produced either enterotoxin A, or enterotoxin B, or both. These findings suggest a possible role for enterotoxins in the pathogenesis of S. aureus disease other than food poisoning.     

Molecular subtyping and characterization of bovine and human Streptococcus agalactiae isolates

Streptococcus agalactiae causes severe invasive disease in humans and mastitis in cattle. Temporally matched bovine milk isolates and clinical human invasive isolates (52 each) collected in New York State over 18 months were characterized by molecular subtyping and phenotypic methods to probe the interspecies transmission potential of this species. EcoRI ribotyping differentiated 17 ribotypes, and DNA sequencing of the housekeeping gene sodA and the putative virulence gene hylB differentiated 7 and 17 allelic types, respectively. Human and bovine isolates were not randomly distributed between ribotypes or hylB and sodA clusters. The combined analysis of all subtyping data allowed the differentiation of 39 clonal groups; 26 groups contained only bovine isolates, and 2 groups contained both human and bovine isolates. The EcoRI ribotype diversity among bovine isolates (Simpson's numerical index of discrimination [mean +/- standard deviation], 0.90 +/- 0.05) being significantly higher than that among human isolates (0.42 +/- 0.15) further supports that these isolates represent distinct populations. Eight human isolates, but no bovine isolates, showed an IS1548 transposon insertion in hylB, which encodes a hyaluronidase. Based on data for 43 representative isolates, human isolates, on average, showed lower hyaluronidase activities than bovine isolates. Isolates with the IS1548 insertion in hylB showed no hyaluronidase activity. Human and bovine isolates did not differ in their abilities to invade HeLa human epithelial cells. Our data show that (i) EcoRI ribotyping, combined with hylB and sodA sequencing, provides a discriminatory subtype analysis of S. agalactiae; (ii) most human invasive and bovine S. agalactiae isolates represent distinct subtypes, suggesting limited interspecies transmission; and (iii) hyaluronidase activity is not required for all human infections.     

Electrophoretic type B2 of carboxylesterase B for characterisation of highly pathogenic Escherichia coli strains from extra-intestinal infections

The frequency of electrophoretic types B1 (fast mobilities) and B2 (slow mobilities) of carboxylesterase B, and alpha-haemolysin and mannose-resistant haemagglutinin (MRHA) production were compared in 705 strains of Escherichia coli isolated from cases of septicaemia, urinary tract infection (UTI) and other extra-intestinal infections from different geographical origins, in particular France, America (USA and Canada) and Oceania (Australia and New Zealand). In all groups of strains, whether classified according to their clinical or their geographical origin, electrophoretic type B2 was phenotypically linked with alpha-haemolysin and MRHA production. Haemolytic type B2 strains were isolated more frequently from France and Oceania than America whereas the proportions demonstrating production of MRHA were similar among the three groups. Type B2 strains were more frequently isolated from UTI and other infections than from septicaemia. This is attributed to the high frequency of immunocompromised subjects in the septicaemia group. Our results establish the suitability of using the type B2 of carboxylesterase B as a molecular marker for highly pathogenic E. coli strains implicated in extra-intestinal infections in man.     

Inactivation of the alpha-haemolysin gene of Staphylococcus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins

S. aureus strain 8325-4 was shown to produce alpha-, beta-, delta- and gamma-haemolysins by haemolytic assays and immunoblotting. Hybridization experiments indicated that a single copy of the alpha-haemolysin gene (hla) resides in the chromosome. Site-directed mutagenesis was used to inactivate the hla gene. This gene, which had previously been cloned in E. coli, was inactivated in vitro by inserting a fragment carrying an erythromycin resistance marker. Shuttle plasmids were constructed and transformed into 8325-4 and non-haemolytic recombinants enriched by a plasmid incompatibility technique. A previously isolated Tn551 insertion defective in alpha-haemolysin was not located in hla. It had pleiotropic defects in expression of alpha-, beta- and delta-haemolysins. Expression of alpha-haemolysin from a plasmid-located hla gene was very low. In contrast, hla-erm mutants were deficient only in alpha-haemolysin and allowed high level expression of the plasmid-borne hla gene. The Tn551 insertion is probably located in a gene encoding a positive regulatory element required for expression of several exoproteins. An hla-erm mutant was less virulent than the otherwise isogenic 8325-4 hla+ strain in a mouse peritonitis model, confirming that alpha-haemolysin is an important virulence factor.     

Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.     

Virulence genes in verocytotoxigenic Escherichia coli strains isolated from humans and cattle

Verocytotoxigenic Escherichia coli (VTEC) causing diarrhoea, haemorrhagic colitis and haemolytic-uremic syndrome usually have additional traits such as the adhesin intimin and a large plasmid that seems to increase virulence. There are, however, isolates of VTEC causing serious symptoms that do not harbour these traits. In the present study we have used PCR with primers detecting adhesin genes other than eaeA, namely fimA, papC, sfaD/sfaE and daaE. We have also used PCR to detect the genes hlyA and iutA that besides the plasmid-borne gene E-hly possibly support the bacterial access to iron. The aim of the study was to identify and compare the presence of virulence genes in VTEC isolates of human and cattle origin. The main finding was that the absence of E-hly might be compensated for by the gene iutA coding for aerobactin or hlyA coding for alpha-haemolysin as 94% of the human VTEC isolates had at least one of these genes. Interestingly, only 45% of VTEC isolated from cattle had any of these genes. We propose that this might be the reason for the relatively low incidence of symptomatic VTEC infections among humans in relation to the high number of VTEC among cattle.     

Comparison of Staphylococcus aureus isolates from bovine and human skin,milking equipment,and bovine milk by phage typing,pulsed-field gel electrophoresis,and binary typing

Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.     

VTEC O157 subtypes associated with the most severe clinical symptoms in humans constitute a minor part of VTEC O157 isolates from Danish cattle

The aim of this study was to compare the distribution of VTEC O157 subtypes isolated from human sporadic infections with those in the Danish bovine reservoir, and to correlate the subtypes with the severity of the clinical symptoms in humans. The study included a total of 149 Danish eae-positive VTEC O157 isolates (63 of bovine origin and 86 from human clinical cases) isolated between 1987 and 2001. All were analysed by vtx-PCR-RFLP and phage typing. The vtx-PCR-RFLP showed that isolates carrying the vtx2 gene was more than four times as prevalent among the human clinical isolates (55%) as compared to the bovine isolates (13%). Furthermore, a significant correlation between the presence of the vtx2 gene and development of haemolytic-uraemic syndrome was found. The 149 isolates encompassed 16 different phage types (PTs). The majority (87%) of the human clinical isolates were identified, as PT2, PT4, PT8 or PT14 while only 46% of the bovine isolates belonged to these PTs. PT8 and PT14 were found at similar rates among bovine (36%) and human isolates (40%). However, the predominant PTs in the human isolates, PT2 (19%) and PT4 (28%), were only identified in 2% and 8%, respectively, of the bovine isolates. All but one PT2 and PT4 isolate carried either vtx2 alone or in combination with vtx2c, whereas none of the PT8 and PT14 isolates carried vtx2. The significant overlap between vtx/phage type combinations in bovine and human clinical isolates indicate that cattle are an important reservoir for human VTEC O157 infections in Denmark. However, the vtx2-carrying isolates, causing the most severe clinical symptoms, constitute only a minor fraction of the isolates from the Danish bovine reservoir.     

Molecular Cloning and Characterization of the afa-7 and afa-8 Gene Clusters Encoding Afimbrial Adhesins in Escherichia coli Strains Associated with Diarrhea or Septicemia in Calves

The afa gene clusters, which encode proteins involved in adhesion to epithelial cells, from Escherichia coli strains associated with urinary and intestinal infections in humans have been characterized. Pathogenic isolates of bovine and porcine origin that possess afa-related sequences have recently been described. We report in this work the cloning and characterization of the afa-7 and afa-8 gene clusters from bovine isolates. Hybridization and sequencing experiments revealed that despite similarity in genetic organization, the afa-7 and afa-8 genes, and the well-characterized afa-3 operon expressed by human-pathogenic isolates, correspond to three different members of the afa family of gene clusters. However, like the afa-3 gene cluster, both the afa-7 and afa-8 gene clusters were found to encode an afimbrial adhesin (AfaE) and an invasin (AfaD). The AfaD peptides encoded by the three gene clusters were only 45% identical, but functional complementation experiments indicated that they belong to the same family of invasins. Hemagglutination and adhesion assays demonstrated that the AfaE-VII and AfaE-VIII adhesins bind to different receptors and that these receptors are not the human decay-accelerating factor recognized to be the receptor of all previously described AfaE adhesins. The AfaE-VIII adhesin is very similar to the M agglutinin of human-uropathogenic strains. We used PCR assays to screen 25 bovine strains for afaD and afaE genes of either the afa-7 or afa-8 gene cluster. The afa-8 gene cluster was highly prevalent in bovine isolates previously reported to carry afa-related sequences (23 of 24 strains), particularly in strains producing cytotoxic necrotizing factors (16 of 16 strains). The location of the afa-8 gene cluster on the plasmids or chromosome of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic E. coli strains.     

Characterisation of drug resistance gene cassettes associated with class 1 integrons in clinical isolates of Escherichia coli from Taiwan, ROC

The presence of class 1 integrons in clinical isolates of Escherichia coli was detected by PCR. Of 104 E. coli isolates from Kaohsiung, 54 (52%) carried class 1 integrons, with inserted DNA regions of 1-3 kb. These integrons were located on plasmids, as demonstrated by Southern hybridisation. DNA sequencing was used to identify the genetic content of the integron-variable regions. Different class 1 integrons contained various numbers, kinds and combinations of gene cassettes within their variable regions. These gene cassettes included those encoding resistance to trimethoprim (dfrIa, dfrV, dfr12 and dfr17), aminoglycosides (aadA1a, aadA2, aadA4 and aadB), chloramphenicol (cmlA), erythromycin (ereA2) and beta-lactams (blaP1). An integron carrying three inserted cassettes - dfr12-orJF-aadA2 - was present in 33 (61%) of the 54 isolates with class 1 integrons. Gene cassettes encoding resistance were expressed phenotypically. The results indicate that class 1 integrons are widespread in clinical E. coli isolates in Taiwan. The types, combinations and frequency of the gene cassettes in integrons may reflect the specific selective pressures to which the isolates were exposed and could provide useful surveillance data for relation to antibiotic usage information.     

Clonality and virulence traits of Escherichia coli associated with haemorrhagic septicaemia in turkeys

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST_p) and STIb (ST_h). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.     

Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: Implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India

Summary.  Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of the VP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with the VP7 from several animal and animal rotavirus-related human G3 strains (Simian SA11; equine ERV316 and FI-14; canine CU-1 and K9; porcine 4F; Feline Cat2 and human HCR3, YO and AU1). Partial nucleotide sequence analysis of the NSP1 gene of J63 showed greatest nt sequence homology (95.9%) to the NSP1 gene allele of the Indian G8 strain, isolated from a diarrheic child, which is likely to have been transmitted directly from cattle and 92.6% homology to that of the bovine G8 strain A5-10 suggesting the likely origin of J63 by gene reassortment between a bovine G8 strain and a G3 animal strain. Prevalence of G10P11 strains in cattle and G10P11 or P11 type reassortant strains in asymptomatic neonates as well as detection of G8P[1] strains in diarrheic children support our hypothesis for bidirectional transmission of rotaviruses between humans and cattle and origin of novel strains catalyzed by the age-old traditions and socio-economic conditions in India. Received October 16, 2000 Accepted July 6, 2001     

Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts

To better understand the emergence and transmission of antibiotic-resistant Streptococcus agalactiae, we compared phenotypic and genotypic characteristics of 52 human and 83 bovine S. agalactiae isolates. Serotypes found among isolates from human hosts included V (48.1%), III (19.2%), Ia and Ib (13.5% each), and II (5.8%). Among isolates from bovine hosts, molecular serotypes III and II were predominant (53 and 14.5%, respectively). Four and 21 different ribotypes were found among human and bovine isolates, respectively. A combination of ribotyping and serotyping showed that two bovine isolates were indistinguishable from human isolates. Resistance to tetracycline and erythromycin was more common among human (84.6% and 26.9%, respectively) than bovine (14.5% and 3.6%, respectively) isolates. tetM was found in all tetracycline-resistant human isolates, while tetO was the predominant resistance gene among bovine isolates. tet genes were found among various ribotypes. ermB, ermTR, and mefA were detected among erythromycin-resistant human isolates, while ermB was the only erythromycin resistance determinant among isolates from bovine hosts. For isolates from human hosts, erythromycin resistance genes appeared to be associated with specific ribotypes. We conclude that (i) human and bovine S. agalactiae isolates represent distinct populations; (ii) human host-associated S. agalactiae subtypes may occasionally be transmitted to bovines; (iii) while emergence of erythromycin and tetracycline resistance appears to largely occur independently among human and bovine isolates, occasional cross-species transfer of resistant strains or transmission of resistance genes between human- and bovine-associated subtypes may occur; and (iv) dissemination of antibiotic-resistant S. agalactiae appears to include both clonal spread of resistant strains as well as horizontal gene transfer.     

Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly amplified polymorphic DNA analysis

Streptococcus agalactiae is considered one of the major causes of bovine intramammary infections. It is also found in the vaginas of women without any apparent clinical symptoms, but reports of neonatal infections, causing significant morbidity, are relatively frequent. The aim of this study was to evaluate the genetic diversity of S. agalactiae strains isolated from bovine milk and from asymptomatic women in Québec, Canada, by randomly amplified polymorphic DNA (RAPD) analysis. A total of 185 bovine isolates and 38 human isolates were first serotyped for capsular polysaccharide by double diffusion in agarose gel (bovine isolates) and coagglutination (human isolates). Strains were then studied by RAPD using 3 primers, designated OPS11, OPB17, and OPB18, which were selected from 12 primers. Thirty-eight percent of bovine isolates and 82% of human isolates could be serotyped. Prevalent serotypes were type III (28%) for bovine isolates and types V (26%) and III (24%) for human isolates. RAPD results showed that, taken together, all isolates (of bovine and human origin) shared 58% similarity. Ninety-four percent of these isolates were clustered in four groups (I, II, III, and IV) with 70% similarity among them. Three clusters, A (48 isolates), B (14 isolates), and C (32 isolates), with 79 to 80% similarity were identified within group IV, whereas the three other groups did not present any clusters. Despite some clustering of human isolates, relatively high diversity was seen among them. Relatively high heterogeneity was observed with the RAPD profiles, not only for field strains belonging to different serotypes but also for those within a given serotype.     

Molecular characterization of Cryptosporidium isolates from human and bovine using 18s rRNA gene in Shahriar county of Tehran, Iran

Cryptosporidiosis is a widespread cause of diarrheal diseases of humans, young calves, and many mammals. Although in recent years molecular investigations on Cryptosporidium have increased, no data are available on Iran in this regard. Two species of Cryptosporidium are known to infect human beings; Cryptosporidium hominis which shows anthroponotic transmission among humans and Cryptosporidium parvum which shows zoonotic transmission between animals and humans. Cryptosporidium oocysts, morphologically identified as C. parvum, were isolated from 24 human and 35 bovine cases in Shahriar (suburb of Tehran, Iran), and genotyped by means of a Nested-polymerase chain reaction/restriction fragment length polymorphism analysis of the 18s rRNA gene. The isolates from bovine samples gave zoonotic or 2 genotype (C. parvum), and DNA profiles of human isolates gave two distinct genotypes, namely an anthroponotic or 1 (C. hominis) and zoonotic genotype or 2 (C. parvum).     

Comparison of antimicrobial resistance genes in nontyphoidal salmonellae of serotypes enteritidis, hadar, and virchow from humans and food-producing animals in England and wales

Isolates of Salmonella enterica serovars Enteritidis (n = 17), Hadar (n = 18), and Virchow (n = 13) from cases of human infection and from food production animals were screened using a miniaturized antimicrobial microarray to determine the number and spectra of resistance genes. Among Enteritidis, the number of genes detected was: animal isolates, mean = 4.6; human isolates, mean = 5.3. Resistance to streptomycin, trimethoprim, and sulfonamides was usually encoded by only one resistance gene in animal isolates, but human isolates often carried more than one gene encoding resistance to the same class of antimicrobial. Among Hadar, the number of genes detected was: animal isolates, mean = 2.0; human strains, mean = 2.6. Resistance to streptomycin was encoded by strA, rather than aadA genes because these were detected in only one human isolate. Among Virchow, the number of genes detected was: animal isolates, mean = 1.6; human isolates, mean = 5.6. As with Enteritidis, human Hadar isolates often carried more than one gene encoding resistance to the same class of antimicrobial. Due to the complexity of routes of transmission of Salmonella spp. from food production animals to humans, full phenotypic and genotypic comparison of resistant isolates is critical in ascertaining the sources of resistant isolates.