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Drug-resistant genes, virulence characteristics, and molecular typing of clindamycin-resistant Streptococcus agalactiae in late pregnancy.

BACKGROUND: Streptococcus agalactiae increases the risk of adverse pregnancy outcomes and neonatal infections. Clindamycin is a key alternative for intrapartum prophylaxis in penicillin-allergic women, but the prevalence of clindamycin-resistant S. agalactiae is increasing, posing a significant clinical challenge. METHODS: A total of 178 strains isolated from tertiary hospitals in Jinan and Qingdao, Shandong Province, China, were characterized using antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, serotyping, and analysis of resistance and virulence genes. RESULTS: All strains were susceptible to penicillin, ampicillin, linezolid, vancomycin, and tigecycline. In contrast, resistance rates to erythromycin, levofloxacin, and tetracycline were 95.5%, 60.1%, and 56.7%, respectively. Six serotypes and 15 sequence types belonging to eight clonal complexes were identified. Notable regional differences were observed. The Ib-ST10-CC12 lineage dominated in Jinan, whereas V-ST529-CC327 was predominant in Qingdao. The resistance gene mreA was ubiquitous (100%), followed by ermB (80.3%). The key virulence genes cylE, hylB, and pavA, were detected in all strains. fbsA (99.4%), the alpha protein family (98.9%), cfb (98.3%), the Pilus Island gene cluster (94.9%), and lmb (92.7%) were also highly prevalent. The two major clindamycin resistance genes, erm and lnuB, exhibited distinctly different enrichment patterns among S. agalactiae clonal complexes, despite a certain overlap in CC19 and CC327. Specifically, erm was significantly enriched in CC12 (serotype Ib), CC19 (III/V), and CC327 (III/V). In contrast, lnuB was predominantly restricted to CC19 and CC327, where it defined a unique phylogenetic subcluster. Significant differences in resistance and virulence gene profiles were observed across different clonal complexes. CONCLUSION: Clindamycin-resistant S. agalactiae in late-pregnancy women in Shandong Province, China exhibits a broad resistance spectrum, diverse molecular types, and significant regional heterogeneity. These findings underscore the need for continued surveillance and region-specific strategies for preventing neonatal S. agalactiae infections.

Humans↗

Presence and characterization of extraintestinal pathogenic Escherichia coli virulence genes in F165-positive E. coli strains isolated from diseased calves and pigs.

The virulence genotype profile and presence of a pathogenicity island(s) (PAI) were studied in 18 strains of F165-positive Escherichia coli originally isolated from diseased calves or piglets. On the basis of their adhesion phenotypes and genotypes, these extraintestinal pathogenic strains were classified into three groups. The F165 fimbrial complex consists of at least two serologically and genetically distinct fimbriae: F165(1) and F165(2). F165(1) is encoded by the foo operon (pap-like), and F165(2) is encoded by fot (sfa related). Strains in group 1 were foo and fot positive, strains in group 2 were foo and afa positive, and strains in group 3 were foo positive only. The strains were tested for the presence of virulence genes found mainly in extraintestinal pathogenic E. coli (ExPEC) strains. Although all the strains were positive for the papA variant encoding F11 fimbriae incD, traT, and papC, the prevalence of virulence genes commonly found in PAIs associated with ExPEC strains was highly variable, with strains of group 2 harboring most of the virulence genes tested. papG allele III was detected in all strains in group 1 and in one strain in group 3. All other strains were negative for the known alleles encoding PapG adhesins. The association of virulence genes with tRNA genes was characterized in these strains by using pulsed-field gel electrophoresis and DNA hybridization. The insertion site of the foo operon was found at the pheU tRNA locus in 16 of the 18 strains and at the selC tRNA locus in the other 2 strains. Furthermore, 8 of the 18 strains harbored a high-pathogenicity island which was inserted in either the asnT or the asnV/U tRNA locus. These results suggest the presence of one or more PAIs in septicemic strains from animals and the association of the foo operon with at least one of these islands. F165-positive strains share certain virulence traits with ExPEC, and most of them are pathogenic in piglets, as tested in experimental infections.

Animals↗

Escherichia coli serotype O15:K52:H1 as a uropathogenic clone.

To clarify the clinical and bacteriological correlates of urinary-tract infection (UTI) due to Escherichia coli O15:K52:H1, during a 1-year surveillance period we prospectively screened all 1, 871 significant E. coli urine isolates at the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, for this serotype and assessed the epidemiological features of community-acquired UTI due to E. coli O15:K52:H1 versus other E. coli serotypes. We also compared the 25 O15:K52:H1 UTI isolates from the present study with 22 O15:K52:H1 isolates from other, diverse geographic locales and with 23 standard control strains (8 strains from the ECOR reference collection and 15 strains of nonpathogenic O:K:H serotypes) with respect to multiple phenotypic and genotypic traits. Although E. coli O15:K52:H1 caused only 1.4% of community-acquired E. coli UTIs during the surveillance period, these UTIs were more likely to present as pyelonephritis and to occur in younger hosts, with similar risk factors, than were UTIs due to other E. coli serotypes. Irrespective of geographic origin, E. coli O15:K52:H1 strains exhibited a comparatively restricted repertoire of distinctive virulence factor profiles (typically, they were positive for papG allele II, papA allele F16, and aer and negative for sfa, afa, hly, and cnf1), biotypes, ribotypes, and amplotypes, consistent with a common clonal origin. In contrast, their antimicrobial resistance profiles were more extensive and more diverse than those of control strains. These findings indicate that E. coli O15:K52:H1 constitutes a broadly distributed and clinically significant uropathogenic clone with fluid antimicrobial resistance capabilities.

Bacteremia↗

Enteroaggregative Escherichia coli from humans and animals differ in major phenotypical traits and virulence genes.

Enteroaggregative Escherichia coli (EAEC) is characterized by the expression of the aggregative adherence pattern to cultured epithelial cells. In this study, we determined the phenotypic and genotypic relationships among 86 EAEC strains of human and animal (calves, piglets and horses) feces. Serotypes and the presence of EAEC virulence markers were determined, and these results were associated with ribotyping. Strains harboring aggR (typical EAEC) of human origin were found carrying several of the searched markers, while atypical EAEC harbored none or a few markers. The strains of animal origin were classified as atypical EAEC (strains lacking aggR) and harbored only irp2 or shf. Strains from humans and animals belonged to several different serotypes, although none of them prevailed. Sixteen ribotypes were determined, and there was no association with virulence genes profiles or serotypes. Relationship was not found among the strains of this study, and the assessed animals may not represent a reservoir of human pathogenic typical EAEC.

Animals↗

Phenotypic and molecular characteristics of Escherichia coli isolated from aquatic environment of Bangladesh.

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.

Bangladesh↗

Heterogeneity of mycolactones produced by clinical isolates of Mycobacterium ulcerans: implications for virulence.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that M. ulcerans isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of M. ulcerans representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that M. ulcerans produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M(+)Na](+) at m/z 765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na](+) at m/z 726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na](+) m/z 781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na](+) m/z 447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.

Animals↗

Variations in lethal toxin and cholesterol-dependent cytolysin production correspond to differences in cytotoxicity among strains of Clostridium sordellii.

Clostridium sordellii is an emerging human pathogen and frequent contaminant of cadaver-derived tissue transplant material. Herein, we provide data suggesting the potential for severe C. sordellii-associated disease may be dictated by whether the specific strain produces lethal toxin (TcsL) or sordellilysin (SDL), a cholesterol-dependent cytolysin. The virulence factor profiles of 14 C. sordellii isolates were determined, and culture supernatant from six of the isolates was found to be cytotoxic to mammalian cells; yet, only one of these strains conferred cytotoxicity via production of TcsL. Cytotoxicity of TcsL- strains correlated with the production of sordellilysin, which was also recognized by an antiperfringolysin O antibody. However, supernatant from TcsL+, SDL- strains demonstrated a lower LD50 relative to TcsL-, SDL+ strains, suggesting the potential for severe C. sordellii-associated disease may be determined by the particular strain colonizing the host.

Animals↗

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida.

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.

Animals↗

Haemolytic Escherichia coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains.

Twenty-four haemolytic Escherichia coli strains were isolated from dogs with diarrhea. The strains were serotyped and analysed by polymerase chain reaction (PCR) for genes encoding virulence factors associated with E. coli that cause diarrhea in animals. Adhesion antigen production was deduced from haemagglutination experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat extracts was also used as an indication for the production of adhesive structures. The majority of the strains was shown to produce this type of virulence factor. Adhesion and invasion tests of the strains and Caco-2 cells showed that all strains adhered and that two were invasive. The two invasive strains were positive in the intimin PCR and one of them also contained genes encoding CS31A. The PCR for heat stable toxin (ST) was positive in only four strains, as was the presence of F17 fimbrial genes. Surprisingly, 19 strains had intact P fimbrial operons, coding for an adhesin involved in urinary tract infection (UTI). The cytotoxic necrotising factor 1 (CNF1) gene, also mainly found in UTI was likewise detected in these 19 strains. Cytolethal distending toxin (Cdt) genes were found in five strains. The high number of strains positive for CNF1 and P fimbriae prompted us to test the strains in a multiplex PCR used to test E. coli isolated from UTI in various species for 30 virulence associated genes. The data showed that the majority of the diarrhea isolates have virulence factor profiles highly similar to UTI E. coli isolates from dogs. This raises the question whether these isolates are real intestinal pathogens or "innocent bystanders". However, since CNF1 producing necrotoxic E. coli (NTEC) strains isolated from humans, pigs and calves with diarrhea appear to be highly related to our strains, it might be that in dogs this type of isolate is capable of causing not only UTI, but also diarrhea. If this is the case and this type of isolate is "bifunctional", domestic animals likely constitute a reservoir of NTEC strains which can be also pathogenic for humans.

Adhesins, Bacterial↗

The prevalence and spread of Escherichia coli O157:H7 at a commercial beef abattoir.

AIMS: To investigate the prevalence and virulence characteristics of Escherichia coli O157:H7 after a number of beef process operations at a commercial Irish abattoir. METHODS AND RESULTS: Two 12-month studies were carried out. The first study (study 1) examined the prevalence of E. coli O157:H7 at up to six sites on carcasses at eight stages of the dressing, washing, chilling and boning process. The second study (study 2) examined the prevalence of E. coli O157:H7 in bovine faeces and rumen contents post-slaughter and on dressed, washed carcasses. Isolates from both studies were phage-typed and the presence of genes encoding verocytotoxin, enterohaemolysin and intimin production was determined. E. coli O157:H7 was isolated from four of 36 carcasses in study 1. E. coli O157:H7 was detected during hide removal and was detected at multiple carcass sites and multiple process stages, including boning. On two carcasses, contamination was first detected at the bung following its freeing and tying. All isolates from study 1 were phage type (PT) 2, eaeAO157 and ehlyA positive, but were verocytotoxin 1 (VT1) and verocytotoxin 2 (VT2) negative. In study 2, E. coli O157:H7 was isolated from 2.4% of faecal, 0.8% of rumen and 3.2% of carcass samples. In some cases, isolates recovered from the faeces of a particular animal, the resulting carcass and adjacent carcasses on the line had the same phage typing and virulence characteristic profile patterns. All isolates from study 2 were eaeAO157 and ehlyA positive and only one isolate was VT1 and VT2 negative. Most isolates were PT 32. A higher frequency of positive isolations was noted from samples taken during spring and late summer. CONCLUSION: These studies show that in a typical Irish beef abattoir, carcass contamination with E. coli O157:H7 can occur during hide removal and bung tying and this contamination can remain on the carcass during subsequent processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data that is necessary for the understanding of how E. coli O157:H7 contamination of beef occurs.

Abattoirs↗

Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a well-recognized cause of bloody diarrhea and hemolytic-uremic syndrome (HUS). Non-O157 STEC contribute to this burden of illness but have been underrecognized as a result of diagnostic limitations and inadequate surveillance. METHODS: Between 1983 and 2002, 43 state public health laboratories submitted 940 human non-O157 STEC isolates from persons with sporadic illnesses to the Centers for Diseases Control and Prevention reference laboratory for confirmation and serotyping. RESULTS: The most common serogroups were O26 (22%), O111 (16%), O103 (12%), O121 (8%), O45 (7%), and O145 (5%). Non-O157 STEC infections were most frequent during the summer and among young persons (median age, 12 years; interquartile range, 3-37 years). Virulence gene profiles were as follows: 61% stx(1) but not stx(2); 22% stx(2) but not stx(1); 17% both stx(1) and stx(2); 84% intimin (eae); and 86% enterohemolysin (E-hly). stx(2) was strongly associated with an increased risk of HUS, and eae was strongly associated with an increased risk of bloody diarrhea. STEC O111 accounted for most cases of HUS and was also the cause of 3 of 7 non-O157 STEC outbreaks reported in the United States. CONCLUSIONS: Non-O157 STEC can cause severe illness that is comparable to the illness caused by STEC O157. Strains that produce Shiga toxin 2 are much more likely to cause HUS than are those that produce Shiga toxin 1 alone. Improving surveillance will more fully elucidate the incidence and pathological spectrum of these emerging agents. These efforts require increased clinical suspicion, improved clinical laboratory isolation, and continued serotyping of isolates in public health laboratories.

Disease Outbreaks↗

Prevalence of childhood diarrhoea-associated Escherichia coli in Thailand.

Escherichia coli isolates (n=2629) were collected between 1996 and 2000 from 2100 Thai children less than 12 years of age with acute diarrhoea. Enterotoxigenic (ETEC), enteroinvasive (EIEC), Shiga-toxin-producing (STEC), enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli were identified by their virulence marker profiles, as determined by multiplex PCR, and HeLa cell-adherence patterns. Serogroups of isolates were determined using 43 monovalent O antisera. Of 2629 isolates, 16.9% were identified as diarrhoeagenic E. coli, and the mean isolation rates per year were 10.2% for EAEC (range 8-12.5%), 3.2% for EPEC (0-8%), 3.0% for ETEC (2-5.4%), 0.5% for EIEC (0-1%) and 0.04 % for STEC (0-0.1%). The isolation rates of pathotypes from four different age groups (0-5 months, 6-11 months, 1-2 years and 2-12 years) in 905 children whose ages were recorded were respectively 19.3, 18.2, 9.1 and 8.1% for EAEC, 3.1, 4.3, 1.7 and 2.2% for EPEC and 2.6, 2.3, 1.3 and 5% for ETEC. About 38% of diarrhoeagenic E. coli, including 55.1, 66.7, 100, 45.9 and 29%, respectively, of ETEC, EIEC, STEC, EPEC and EAEC, and 24% of non-diarrhoeagenic E. coli were O-antigen typable. Only four serogroups (9.3%) were restricted to single pathotypes, whereas 27 serogroups (62.8%) were not restricted to any pathotype. This study shows that EAEC are the most prevalent diarrhoea-associated pathotype in Thai children.

Acute Disease↗

Dynamics and virulence of Enterobacteriaceae reservoirs harboring blaCTX-M group 1 in community wastewater.

UNLABELLED: Extended-spectrum beta-lactamase (ESBL)-producing bacteria are ubiquitous and can cause serious infections. Here, we examined untreated community wastewater influent as a reservoir for blaCTX-M group 1 organisms and their virulence potential. Raw influent samples (n = 268) were collected from four wastewater treatment plants (WWTPs) representing dense urban populations. We observed that blaCTX-M group 1 levels were high at all WWTPs and only ~1-2 log10 lower and not correlated to common human-specific microbiome fecal markers, Lachno3 and HF183, indicating a lack of connection to human fecal inputs. Concentrations of blaCTX-M group 1 genes and markers for presumptive host organisms Escherichia coli and Klebsiella pneumoniae were influenced by travel time and season. Amplicon sequencing revealed high diversity of blaCTX-M group 1-9 genes, with 63% belonging to group 1. Selective culture and 16S rRNA gene sequencing showed blaCTX-M group 1 isolates were 26% E. coli, 26% K. pneumoniae, 40% other Enterobacteriaceae, and 8% Aeromonas. Overall, E. coli averaged 3.6E7 cells/L, with 3% of all E. coli found to contain blaCTX-M group 1. Whole-genome sequencing of blaCTX-M group 1 E. coli from wastewater revealed resistance and virulence gene profiles similar to clinical isolates and distinct from other wastewater ESBL-resistant and non-resistant E. coli. Interpretation of wastewater data needs to consider both the existence of environmental reservoirs that contain potentially pathogenic organisms and the strong influence the dynamics of the conveyance system can have on final concentrations measured at the WWTP. IMPORTANCE: The CTX-M enzyme family is highly abundant in nosocomial, community, and environmental settings and is leading to treatment of infections with carbapenem antibiotics, a last-line therapeutic option. The progressive increase of the clinically relevant blaCTX-M group 1 resistance genes in the human population warrants investigation, particularly to understand the establishment and dynamics of environmental reservoirs. This study utilized molecular and culture methods to gain insight into the possible origin, abundance, and dynamics of blaCTX-M group 1 genes in untreated wastewater influent samples. We found extremely high levels of these genes, with Escherichia coli as a major host organism that closely resembled clinical strains, suggesting they are seeded and propagate in sewer pipe systems. The significance of our research is in developing approaches to monitor antimicrobial resistance reservoirs in community wastewater, which could shed light on global burdens and potential transmission cycles and indicate increasing inputs of clinically relevant strains originating from human populations.

E. coli↗

Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model.

Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingivalis, a bacterium implicated as a major etiological agent of chronic periodontitis. Three genes. rgpA, rgpB, and kgp, encode an Arg-x-specific proteinase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x-specific proteinase and adhesins (Kgp), respectively. The contribution to pathogenicity of each of the proteinase genes of P. gingivalis W50 was investigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp. Whole-cell Arg-x-specific proteolytic activity of both the RgpA(-) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) relative to that of the wild-type W50. However, for the Kgp(-) isogenic mutant, whole-cell Arg-x activity was similar to that of the wild-type strain. Whole-cell Lys-x proteolytic activity of the RgpA(-) and RgpB(-) mutants was not significantly different from that of wild-type W50, whereas the Kgp(-) mutant was devoid of Lys-x whole-cell proteolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using proteinase-specific antibodies of cell sonicates of the wild-type and mutant strains showed that the proteinase catalytic domain of each of the mutants was not expressed. This analysis further showed that RgpB appeared as 72- and 80-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processed 45-kDa and 48-kDa bands, respectively. In the murine lesion model, mice were challenged with three doses of each mutant and wild-type strain. At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each of the mutants, whereas wild-type W50 induced large ulcerative lesions. At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-type strain died, whereas mice challenged with the RgpA(-) and RgpB(-) isogenic mutants did not die but developed lesions. Mice challenged with the Kgp(-) isogenic mutant at this dose did not develop lesions. At a 1.2 x 10(10) viable-cell dose, only 40% of mice challenged with the Kgp(-) mutant developed lesions, and these lesions were significantly smaller than lesions induced by the wild-type strain at the 3.0 x 10(9) viable-cell dose. All the mice challenged with the RgpA(-) mutant died at the 1.2 x 10(10) viable-cell dose, whereas only 20% died when challenged with the RgpB(-) mutant at this dose. Wild-type phenotype was restored to the RgpB(-) mutant by complementation with plasmid pNJR12::rgpB containing the rgpB gene. There was no difference between the pNJR12::rgpB-complemented RgpB(-) mutant and the wild-type W50 strain in whole-cell Arg-x activity, protein profile, or virulence in the murine lesion model. These results show that the three proteinases, RgpA, RgpB, and Kgp, all contributed to virulence of P. gingivalis W50 in the murine lesion model and that the order in which they contributed was Kgp >> RgpB > or = RgpA.

Adhesins, Bacterial↗

Molecular characterization of the Prospect Hill virus M RNA segment: a comparison with the M RNA segments of other hantaviruses.

Complementary DNA representing the genomic M RNA segment of the Prospect Hill (PH) Hantavirus was cloned and its nucleotide sequence determined. The PH virus M RNA segment is 3707 nucleotides in length and has a long open reading frame in the viral complementary-sense RNA with a coding capacity of 1142 amino acids. The predicted gene product of the PH virus M segment was compared with the corresponding gene products of Hantaan virus strain 76-118 (Hantaan), Sapporo rat virus strain SR-11 (SR) and Puumala virus strain Hällnäs B1 (Hällnäs). The amino acid sequence identities between the G1 and G2 proteins of PH virus and Hällnäs virus are respectively 74% and 79%. In contrast, the amino acid sequence similarities between the G1 proteins of PH virus and SR virus or Hantaan virus are only 50%. However the G2 proteins of SR and Hantaan viruses were more closely related to the G2 protein of PH virus with amino acid sequence similarities of approximately 62%. The G1 proteins of all four viruses had three potential asparagine-linked glycosylation sites conserved and there was one conserved site in the G2 proteins. Hydrophilicity plots of the four virus glycoproteins were very similar. The region of greatest hydrophilicity was conserved in the Hällnäs, SR and Hantaan viruses, and was located near the C terminus of the G1 protein, whereas the region of greatest hydrophilicity in the PH virus glycoprotein precursor is located closer to the N terminus of the G1 protein. Our data demonstrate that despite differences in the serotypic profiles and virulence of PH and Hällnäs viruses, their G1 and G2 proteins are closely related. We conclude that PH and Hällnäs viruses may have evolved along a separate evolutionary pathway in the Hantavirus genus from that of SR and Hantaan viruses.

Amino Acid Sequence↗

Safety assessment of the oral cavity probiotic Streptococcus salivarius K12.

Streptococcus salivarius is a prominent member of the oral microbiota and has excellent potential for use as a probiotic targeting the oral cavity. In this report we document safety data relating to S. salivarius K12, including assessment of its antibiogram, metabolic profiles, and virulence determinants, and we examine the microbial composition of saliva following the dosing of subjects with K12.

Anti-Bacterial Agents↗

Rapid identification and differentiation of clinical isolates of enteropathogenic Escherichia coli (EPEC), atypical EPEC, and Shiga toxin-producing Escherichia coli by a one-step multiplex PCR method.

Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic E. coli, and Shiga toxin-producing E. coli differ in their virulence factor profiles, clinical manifestations, and prognosis, and they require different therapeutic measures. We developed and evaluated a robust multiplex PCR to identify these pathogroups based on sequences complementary to escV, bfpB, stx1, and stx2.

DNA, Bacterial↗

Molecular characterization of Salmonella enterica subsp. enterica serovar choleraesuis field isolates and differentiation from homologous live vaccine strains suisaloral and SC-54.

Four independent molecular methods were used to characterize the Salmonella enterica subsp. enterica serovar choleraesuis live vaccine strains SC-54 and Suisaloral and to differentiate them from S. choleraesuis field isolates. Plasmid analysis revealed the presence of seven plasmid profiles. A virulence plasmid of 52-kbp was identified by hybridization with an spvB-spvC gene probe in each of the S. choleraesuis field isolates and in the Suisaloral vaccine strain, but not in the SC-54 vaccine strain. Ribotyping, performed with a gene probe that recognized 23S, 16S, and 5S rRNA genes, resulted in three closely related hybridization patterns. IS200 elements were not detected in the field isolates or in the two S. choleraesuis live vaccine strains. Macrorestriction analysis with the enzymes XbaI, SpeI, NotI, and SfiI differentiated the 29 S. choleraesuis strains included in this study into 10, 13, 8, and 13 different fragment patterns, respectively. While the Suisaloral vaccine strain showed a unique XbaI macrorestriction pattern, the fragment patterns of the SC-54 strain obtained with the different enzymes were shared by 2 to 18 S. choleraesuis field strains. A combination of plasmid analysis and macrorestriction analysis proved to be most suitable for the molecular typing of S. choleraesuis and the differentiation of both live vaccine strains from field isolates of this serovar.

Animals↗