Serological Characterization of Haemophilus Pleuropneumoniae (Actinobacillus Pleuropneumoniae) Strains and Proposal of a New Serotype: Serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain. In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses. In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Experimental Infection of Newborn Piglets and Weanling Pigs with Haemolytic E. Coli,Strain A1, Serotype 0149:K91:H10

The first outbreak in Denmark of enteritis in newborn piglets due to infection with E. coli serotype 0149:K91:H10 was demonstrated in the autumn 1966 (Knox & Dam 1970). Since then this serotype has spread rapidly and is to-day responsible for the vast majority of cases of enteric E. eoli infection in newborn piglets, while among pigs at weaning age it occurs with much the same frequency as haemolytic E. coli serotypes 08: K87, 0138:K81, and 0141:K85 ab.     

Differences in the population structure of invasive Streptococcus suis strains isolated from pigs and from humans in The Netherlands

Streptococcus suis serotype 2 is the main cause of zoonotic S. suis infection despite the fact that other serotypes are frequently isolated from diseased pigs. Studies comparing concurrent invasive human and pig isolates from a single geographical location are lacking. We compared the population structures of invasive S. suis strains isolated between 1986 and 2008 from human patients (N?=?24) and from pigs with invasive disease (N?=?124) in The Netherlands by serotyping and multi locus sequence typing (MLST). Fifty-six percent of pig isolates were of serotype 9 belonging to 15 clonal complexes (CCs) or singleton sequence types (ST). In contrast, all human isolates were of serotype 2 and belonged to two non-overlapping clonal complexes CC1 (58%) and CC20 (42%). The proportion of serotype 2 isolates among S. suis strains isolated from humans was significantly higher than among strains isolated from pigs (24/24 vs. 29/124; P<0.0001). This difference remained significant when only strains within CC1 and CC20 were considered (24/24 vs. 27/37,P?=?0.004). The Simpson diversity index of the S. suis population isolated from humans (0.598) was smaller than of the population isolated from pigs (0.765, P?=?0.05) indicating that the S. suis population isolated from infected pigs was more diverse than the S. suis population isolated from human patients. S. suis serotype 2 strains of CC20 were all negative in a PCR for detection of genes encoding extracellular protein factor (EF) variants. These data indicate that the polysaccharide capsule is an important correlate of human S. suis infection, irrespective of the ST and EF encoding gene type of S. suis strains.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.     

Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.     

Population Structure and Antimicrobial Resistance Profiles of Streptococcus suis Serotype 2 Sequence Type 25 Strains

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent.     

Suicin 3908, a New Lantibiotic Produced by a Strain of Streptococcus suis Serotype 2 Isolated from a Healthy Carrier Pig

While Streptococcus suis serotype 2 is known to cause severe infections in pigs, it can also be isolated from the tonsils of healthy animals that do not develop infections. We hypothesized that S. suis strains in healthy carrier pigs may have the ability to produce bacteriocins, which may contribute to preventing infections by pathogenic S. suis strains. Two of ten S. suis serotype 2 strains isolated from healthy carrier pigs exhibited antibacterial activity against pathogenic S. suis isolates. The bacteriocin produced by S. suis 3908 was purified to homogeneity using a three-step procedure: ammonium sulfate precipitation, cationic exchange HPLC, and reversed-phase HPLC. The bacteriocin, called suicin 3908, had a low molecular mass; was resistant to heat, pH, and protease treatments; and possessed membrane permeabilization activity. Additive effects were obtained when suicin 3908 was used in combination with penicillin G or amoxicillin. The amino acid sequence of suicin 3908 suggested that it is lantibiotic-related and made it possible to identify a bacteriocin locus in the genome of S. suis D12. The putative gene cluster involved in suicin production by S. suis 3908 was amplified by PCR, and the sequence analysis revealed the presence of nine open reading frames (ORFs), including the structural gene and those required for the modification of amino acids, export, regulation, and immunity. Suicin 3908, which is encoded by the suiA gene, exhibited approximately 50% identity with bovicin HJ50 (Streptococcus bovis), thermophilin 1277 (Streptococcus thermophilus), and macedovicin (Streptococcus macedonicus). Given that S. suis 3908 cannot cause infections in animal models, that it is susceptible to conventional antibiotics, and that it produces a bacteriocin with antibacterial activity against all pathogenic S. suis strains tested, it could potentially be used to prevent infections and to reduce antibiotic use by the swine industry.     

Actinobacillus Pleuropneumoniae Serotype 5, Subtypes a and b: Cross Protection Experiments

Vaccination of pigs with a killed culture of A. pleuropneumoniae serotype 5, strain K17 (subtype a) afforded a high degree of protection against challenge with strains L20 and T928 (subtype b). The reverse experiment showed that strain L20 gave good protection against challenge with strain K17 whereas strain T928 did not afford an acceptable protection against challenge with this strain. The considerable cross immunity shown to exist between strains K17 and L20 indicates a high degree of homogeneity of the antigenic determinants of the two strains involved in induction of protective immunity and suggest that antibodies to capsular subtype specific determinants may not play a significant role in the specific defence against A. pleuropneumoniae strains belonging to serotype 5. The finding that a vaccine prepared from strain T928 did not afford an acceptable protection against challenge with strain K17 indicates a variable expression among serotype 5 strains of the antigenic determinants which induce protective immunity against A. pleuropneumoniae infection.     

Effect of Spatial Separation of Pigs on Spread of Streptococcus suis Serotype 9

The spread of an infectious agent in a population can be reduced by interfering in the infectiousness or susceptibility of individuals, and/or in their contact structure. The aim of this study was to quantify the effect of prevention of direct contact between infectious and susceptible pigs on the transmission of Streptococcus suis (S. suis). In three replicate experiments, S. suis-free pigs were housed in boxes either in pairs (25 pairs) or alone (15 pigs). The distance between the boxes was ±1 m. At 7 weeks of age, one pig of each pair was inoculated intranasally with S. suis serotype 9; the other pigs were exposed to S. suis by either direct (pairs) or indirect contact (individually housed pigs). Tonsillar brush and saliva swab samples from all pigs were collected regularly for 4 weeks post inoculation to monitor colonization with S. suis. All inoculated pigs became infected, and their pen mates became colonized within 2 days. Thirteen indirectly exposed pigs became positive within 7–25 days after exposure. The rate of direct transmission β_dir was estimated to be 3.58 per pig per day (95% CI: 2.29–5.60). The rate of indirect transmission increased in time, depending on the cumulative number of days pigs tested positive for the presence of S. suis. The estimate β’_ind was 0.001 (95% CI: 0.0006–0.0017) new infections per pig per day for each day that an infected pig was tested positive for S. suis. We conclude that prevention of direct contact reduces the rate at which susceptible pigs become colonized. Simulation studies using these parameters showed, however, that such intervention measure would not limit S. suis serotype 9 spread in a commercial pig farm to a relevant extent, implying that spatial separation of groups op pigs within a compartment would not be effective on a farm.     

Isolation of sucrose-negativeYersinia enterocolitica biotype 3 serotype 03 strains and their pathogenicity

The sucrose-negative strains ofYersinia enterocolitica biotype 3 serotype 03 phage type 2 were isolated from cecal contents and oral cavity swabs of slaughtered pigs and from a swab of a skinner at the slaughterhouse. These organisms differed fromY. kristensenii, determined by assaying the antibiotic susceptibilities to ampicillin, carbenicillin, and cephalothin. These organisms showed positive reactions in the presence of 44 Md plasmid, a calcium dependency, autoagglutination activity, and produced diarrhea in mice and a negative reaction for pyrazinamidase activity. Plasmid digestion with restriction endonucleases isolated from this organism showed the same patterns in biotypes 3 and 4 serotype 03. Therefore, the sucrose-negative strains ofY. enterocolitica biotype 3 serotype 03 are apparently pathogenic.     

Studies on mycobacteria isolated from animals, with special reference to the agglutination test

Ninety-three strains of slowly-growing mycobacteria were studied biochemically. Ninety of these were isolated from animals (pigs, cattle, dog and poultry) and three from dust and sawdust-bedding in a pighouse. One strain from a lymph node of a pig was identified as M. gordonae. Ninety-two strains fitted into the M. aviam-intracellulare complex. Of the 92 biochemically confirmed M. avium-intracellulare strains, 78 were tested serologically ad modum Schaefer. Of 73 strains from pigs, one was serotype 1, fifty serotype 2 and eight serotype 8, while two could not be type and twelve were autoagglutinable. Three strains from pighouse environment were serotype 8 and two from cattle and a dog were both serotype 2. A slight modification of Schaefer's agglutination method, using smaller amounts of antigen and antiserum, was developed.     

合肥地区动物源性大肠埃希菌的血清型分布与耐药性分析

目的了解安徽省合肥地区动物源性大肠埃希菌的血清型分布和耐药状况,以期筛选出菌苗株和指导临床合理用药。方法对46份疑似大肠埃希菌病病料进行细菌分离培养、生化编码鉴定和致病性测定。采用玻片凝集试验对分离到的46株致病性大肠埃希菌进行血清型鉴定。同时分别采用K-B纸片琼脂扩散法和双纸片增效法检测致病性大肠埃希菌的耐药性和ESBLs阳性菌株。结果46株致病性大肠埃希菌中,除7株细菌未能定型外,其余39株细菌分布于10个血清型,O127：K63血清型为优势血清型,占定型菌株的33．33％。46株致病性大肠埃希菌对21种抗菌药物均呈现不同程度的耐药性,15个ESBLs阳性菌株表现为多重耐药,对各种抗菌药物的耐药率均高于ESBLs阴性菌株。结论O127：K63血清型为优势血清型,可作为菌苗株。合肥地区动物源性大肠埃希菌耐药性较为严重,尤其是产ESBLs大肠埃希菌多重耐药更为突出。     

Characterization of Bacillus thuringiensis ser. balearica (Serotype H48) and ser. navarrensis (Serotype H50): Two Novel Serovars Isolated in Spain

The novel strains of Bacillus thuringiensis PM9 and NA69, isolated from soil samples in Spain, were classified and characterized in terms of their crystal proteins, plasmid profile, cry genes content, and their toxicological properties against several species of Lepidoptera, Coleoptera, and Diptera. Both strains share morphological and biochemical characteristics with previously described B. thuringiensis strains, although their unique H antigens identify them as two new serotypes. Two new serovar names, B. thuringiensis serovar balearica (H serotype 48) and B. thuringiensis serovar navarrensis (H serotype 50) are proposed for the type strains PM9 and NA69, respectively. Received: 22 June 1999 / Accepted: 2 August 1999     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.Shigella flexneri is a pathovar of Escherichia coli that is the main causative agent of endemic bacillary dysentery (shigellosis). It is estimated that S. flexneri is responsible for approximately 100 million shigellosis cases annually, resulting in hundreds of thousands of deaths, predominantly in young children (11). Currently no vaccine is available, although there is evidence to suggest that serotype-specific immunity occurs following infection and that induction of immunity can be replicated with vaccines (9). Shigella serotype diversity arises due to differences in the chemical structure of the O-antigen repeating unit in the lipopolysaccharide, which is the main target of the adaptive host immune response following infection.Because immunity to S. flexneri can be conferred by the induction of antibodies directed against the O antigen, an understanding of the prevalence of different serotypes and the underlying basis of serotype diversity can inform appropriate vaccine design. All S. flexneri serotypes (with the exception of serotype 6) share a common O-antigen backbone, consisting of a repeating tetrasaccharide unit that is comprised of one N-acetylglucosamine residue (GlcNAc) and three rhamnose residues (RhaI, RhaII, and RhaIII) (14). The 12 traditionally recognized S. flexneri serotypes differ by the presence or absence of just six different chemical modifications (glucosylations or O acetylations) of the O antigen. The genes responsible for these O-antigen modifications are introduced into the bacterial genome via bacteriophages (3). Glucosylation of the S. flexneri O antigen is mediated by three genes [gtrA, gtrB, and gtr(type)] that are arranged in a single operon known as a gtr cluster. gtrA and gtrB are highly conserved between different gtr clusters and encode proteins involved in transferring the glucosyl group from the cytoplasm into the periplasm, where O-antigen modification is thought to take place. gtr(type) is unique to each gtr cluster and encodes a glucosyltransferase that is responsible for attaching the glucosyl group to a specific sugar unit of the O antigen via a specific linkage (3).Investigations of S. flexneri have typically focused on serotypes for which commercially available typing sera are available. More recently, it has become clear that other serotypes are also epidemiologically important. In Bangladesh in the late 1980s, two novel S. flexneri strains that did not agglutinate with antibodies specific for the traditionally recognized serotypes were isolated (4). Chemical analysis of the O antigen revealed that these strains belonged to a new serotype, which was named serotype 1c due to the similarity its O antigen shares with the O antigens of serotype 1a and 1b strains (19). Serotype 1c has since been isolated in Egypt, Indonesia, Pakistan, and Vietnam (6, 15, 18). Serotype 1c was shown to be the most prevalent S. flexneri serotype in a northern province of Vietnam, accounting for more than a third of all S. flexneri strains isolated from 1998 to 1999 (15). Identification of serotype 1c currently relies on agglutination testing using monoclonal antibody MASF Ic (19).The O antigen of serotype 1c is distinguished by the presence of a disaccharide (two glucosyl groups) linked to the GlcNAc in the tetrasaccharide repeating unit of the O antigen. The first glucosyl group is joined to GlcNAc via an α1→4 linkage, as occurs in the O antigen of serotype 1a and serotype 1b strains (type I modification). The O antigen of serotype 1c is distinguished by the presence of a second glucosyl group that is linked to the first via an α1→2 linkage (Fig. ​(Fig.1).1). Type Ia modification is prerequisite to type Ic modification.Open in a separate windowFIG. 1.Chemical structure of the tetrasaccharide repeat units in the O antigens of S. flexneri serotypes 1a and 1c. Note that the O antigen of serotype 1b (not shown) differs from that of serotype 1a by the O acetylation of l-RhaIII.In this study, the genetic basis of O-antigen modification in serotype 1c was elucidated. Serotype 1c strains isolated from different locations and times were compared to gain insight into the evolution of this serotype. This is the first report of the identification of a glucosyltransferase gene that is responsible for addition of the second glucosyl group, causing serotype conversion from serotype 1a to serotype 1c.     

浙江某市屠宰场猪链球菌血清型、耐药及致病特征

【目的】猪链球菌(Streptococcus suis)是猪的重要病原菌,同时也是人畜共患病原。猪的扁桃体是猪链球菌主要定殖部位之一,是易感猪和人的重要传染源。因此,对屠宰场健康猪进行猪链球菌流行病学调查,具有重要的公共卫生学意义。【方法】本研究自2020年至2021年,从浙江某市屠宰场采集健康猪扁桃体样品,分离鉴定猪链球菌,采用血清型特异性PCR法分型,通过耐药基因检测、药敏试验、斑马鱼毒力实验分析其耐药及致病特征。【结果】131份健康猪扁桃体样品猪链球菌阳性率为62.59%(82/131),共分离猪链球菌68株,其中16型分离率最高,占比16.18%(11/68),其次为31型(11.76%,8/68)、9型(7.35%,5/68)、3型(7.35%,5/68)等。含2种及以上血清型的扁桃体样品占15.85%(13/82)。药敏试验表明,分离株主要对林可酰胺类(100%,68/68)、大环内酯类(98.53%,67/68)、四环素类(100%,68/68)抗生素耐药,所有菌株均属于多药耐药。值得关注的是,有18株菌对青霉素耐药、3株菌对头孢噻肟耐药、2株菌对利福平耐药、11株菌对利...     

Genome Sequence of Aggregatibacter actinomycetemcomitans Serotype c Strain D11S-1

Aggregatibacter actinomycetemcomitans is a major etiological agent of periodontitis. Here we report the complete genome sequence of serotype c strain D11S-1, which was recovered from the subgingival plaque of a patient diagnosed with generalized aggressive periodontitis.Aggregatibacter actinomycetemcomitans is a major etiologic agent of human periodontal disease, in particular aggressive periodontitis (12). The natural population of A. actinomycetemcomitans is clonal (7). Six A. actinomycetemcomitans serotypes are distinguished based on the structural and serological characteristics of the O antigen of LPS (6, 7). Three of the serotypes (a, b, and c) comprise >80% of all strains, and each serotype represents a distinct clonal lineage (1, 6, 7). Serotype c strain D11S-1 was cultured from a subgingival plaque sample of a patient diagnosed with generalized aggressive periodontitis. The complete genome sequencing of the strain was determined by 454 pyrosequencing (10), which achieved 25× coverage. Assembly was performed using the Newbler assembler (454, Branford, CT) and generated 199 large contigs, with 99.3% of the bases having a quality score of 40 and above. The contigs were aligned with the genome of the sequenced serotype b strain HK1651 (http://www.genome.ou.edu/act.html) using software written in house. The putative contig gaps were then closed by primer walking and sequencing of PCR products over the gaps. The final genome assembly was further confirmed by comparison of an in silico NcoI restriction map to the experimental map generated by optical mapping (8). The genome structure of the D11S-1 strain was compared to that of the sequenced strain HK1651 using the program MAUVE (2, 3). The automated annotation was done using a protocol similar to the annotation engine service at The Institute for Genomic Research/J. Craig Venter Institute with some local modifications. Briefly, protein-coding genes were identified using Glimmer3 (4). Each protein sequence was then annotated by comparing to the GenBank nonredundant protein database. BLAST-Extend-Repraze was applied to the predicted genes to identify genes that might have been truncated due to a frameshift mutation or premature stop codon. tRNA and rRNA genes were identified by using tRNAScan-SE (9) and a similarity search to our in-house RNA database, respectively.The D11S-1 circular genome contains 2,105,764 nucleotides, a GC content of 44.55%, 2,134 predicted coding sequences, and 54 tRNA and 19 rRNA genes (see additional data at http://expression.washington.edu/bumgarnerlab/publications.php). The distribution of predicted genes based on functional categories was similar between D11S-1 and HK1651 (http://expression.washington.edu/bumgarnerlab/publications.php). One hundred six and 86 coding sequences were unique to strain D11S-1 and HK1651, respectively (http://expression.washington.edu/bumgarnerlab/publications.php). Genomic islands were identified based on annotations for strain HK1651 and based on manual inspection of contiguous D11S-1 specific DNA regions with G+C bias (http://expression.washington.edu/bumgarnerlab/publications.php). Among 12 identified genomics islands, 5 (B, C, D, E and G; cytolethal distending toxin gene cluster, tight adherence gene cluster, O-antigen biosynthesis and transport gene cluster, leukotoxin gene cluster, and lipoligosaccharide biosynthesis enzyme gene, respectively) correspond to islands 2 to 5 and 8 of strain HK1651 (http://www.oralgen.lanl.gov/) (5). Island F (∼5 kb) is homologous to a portion of the 12.5-kb island 7 in HK1651. Five genomic islands (H to L) were unique to strain D11S-1. The remaining island (A) is a fusion of genomic islands 1 and 6, in strain HK1651. The genome of D11S-1 is largely in synteny with the genome of the sequenced serotype b strain HK1651 but contained several large-scale genomic rearrangements.Strain D11S-1 harbors a 43-kb bacteriophage and two plasmids of 31 and 23 kb (http://expression.washington.edu/bumgarnerlab/publications.php). Excluding an ∼9-kb region of low homology, the phage showed >90% nucleotide sequence identity with AaΦ23 (11). A 49-bp attB site (11) was identified at coordinates 2,024,825 to 2,024,873. The location of the inserted phage was identified in the optical map of strain D11S-1 and further confirmed by PCR amplification and sequencing of the regions flanking the insertion site. A closed circular form of the phage was also detected in strain D11S-1 by PCR analysis of the phage ends. The 23-kb plasmid is homologous to pVT745 (92% nucleotide identities). The 31-kb plasmid is a novel plasmid. It has significant homologies in short regions (<2 kb) to Haemophilus influenzae biotype aegyptius plasmid pF1947 and other plasmids.     

Identification of Recombination in the Envelope Gene of Simian Foamy Virus Serotype 2 Isolated from Macaca cyclopis

The full-length sequence of simian foamy virus serotype 2 (SFVmcy-2), isolated from a Taiwanese macaque, was determined. SFVmcy-2 was highly related to SFV serotype 1 (SFVmcy-1), an isolate from the same species, except in the putative receptor binding domain (RBD) in env, which contained novel sequences related to SFV serotype 3 (SFVagm-3), isolated from an African green monkey. The results identify a potential region of neutralization in SFVs and demonstrate recombination between genetically divergent foamy viruses.     

Synergistic Effects of Sodium Chloride,Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains

Biofilm formation by Listeria monocytogenes is generally associated with its persistence in the food-processing environment. Serotype 1/2a strains make up more than 50% of the total isolates recovered from food and the environment, while serotype 4b strains are most often associated with major outbreaks of human listeriosis. Using a microplate assay with crystal violet staining, we examined biofilm formation by 18 strains of each serotype in tryptic soy broth with varying concentrations of glucose (from 0.25% to 10.0%, wt/vol), sodium chloride (from 0.5% to 7.0%, wt/vol) and ethanol (from 1% to 5.0%, vol/vol), and at different temperatures (22.5°C, 30°C, and 37°C). A synergistic effect on biofilm formation was observed for glucose, sodium chloride, and temperature. The serotype 1/2a strains generally formed higher-density biofilms than the 4b strains under most conditions tested. Interestingly, most serotype 4b strains had a higher growth rate than the 1/2a strains, suggesting that the growth rate may not be directly related to the capacity for biofilm formation. Crystal violet was found to stain both bacterial cells and biofilm matrix material. The enhancement in biofilm formation by environmental factors was apparently due to the production of extracellular polymeric substances instead of the accumulation of viable biofilm cells.Listeria monocytogenes, a Gram-positive bacterium, is capable of causing severe food-borne infections in both humans and animals. The organism is ubiquitous in the environment and can grow in a wide variety of foods, including those stored at refrigeration temperatures. It is particularly difficult to eliminate this bacterium from ready-to-eat foods and food-processing equipment (19). The ability to form biofilms protects the bacterium from stresses in food-processing environments (13, 25). Among the 13 different serotypes described, serotypes 1/2a, 1/2b, and 4b are involved in the majority of human cases of listeriosis. Serotype 4b strains have accounted for most human outbreaks, whereas the majority of L. monocytogenes strains isolated from foods or food-processing plants belong to serotype 1/2a (19).Comparative studies to link the phenotypic attributes of L. monocytogenes strains to serotypes have obtained variable results. Buncic et al. (4) have shown that serotype 1/2a isolates were more resistant to antilisterial bacteriocins than serotype 4b strains at 4°C. They also found that 4b isolates exhibited greater resistance to heat treatments at 60°C and were easier to recover than 1/2a strains immediately following cold storage. Bruhn et al. (3) observed that 1/2a strains (lineage II) grew faster than 4b and 1/2b (lineage I) strains in commonly used enrichment broth media (University of Vermont media I and II). However, other studies have indicated that similar differences could not be linked to a serotype (14), and sequencing results have shown a syntenic relationship between strains of the two serotypes (27).Some L. monocytogenes strains have consistently been isolated from food-processing plants over many years (1, 28). Although several studies have been carried out to identify differences in cell adherence and biofilm formation among different serotypes, conflicting results were obtained. Lineage I isolates (including serotypes 4b, 1/2b, 3c, and 3b) were found to produce higher-density biofilms than lineage II isolates (including serotypes 1/2a, 1/2c, and 3a) (8, 28). However, this conclusion was not supported by other studies (1, 7, 18). For serotype 4b strains, the capacity to form biofilms was reduced when the nutrient level in a medium decreased, while serotype 1/2a strains were not similarly affected (11).It has been suggested that the formation of a biofilm is a stress response by bacterial cells (15, 16). Biofilm research under laboratory conditions may not reflect biofilm formation in the environment. To investigate the behavior of L. monocytogenes in biofilms, a simulated food-processing (SFP) system including several stresses was designed (30). The SFP system was used to study 1/2a and 4b strains in mixed-culture biofilms (31). Bacterial cells from a 1/2a cocktail predominated over 4b strains when exposed to the SFP system for 4 weeks, but no competitive inhibition was observed. Environmental factors, including temperature, sugar, salt, pH, and nutrients that are common in foods and food-processing environments, have been demonstrated to have impacts on L. monocytogenes adhesion and biofilm formation (25). The objectives of this study were to investigate and compare biofilm formation between L. monocytogenes serotype 1/2a strains and serotype 4b strains under a variety of environmental conditions, including different temperatures and varying concentrations of salt, sugar, and ethanol, and to examine the synergistic effects of these factors on biofilm formation by both serotypes.     

Phenotypic and molecular typing of Salmonella strains reveals different contamination sources in two commercial pig slaughterhouses

This study aimed to define the origin of Salmonella contamination on swine carcasses and the distribution of Salmonella serotypes in two commercial slaughterhouses during normal activity. Salmonellae were isolated from carcasses, from colons and mesenteric lymph nodes of individual pigs, and from the slaughterhouse environment. All strains were serotyped; Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Derby isolates were additionally typed beyond the serotype level by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiling (ARP); and a subset of 31 serotype Typhimurium strains were additionally phage typed. PFGE and ARP had the same discriminative possibility. Phage typing in combination with PFGE could give extra information for some strains. In one slaughterhouse, 21% of the carcasses were contaminated, reflecting a correlation with the delivery of infected pigs. Carcass contamination did not result only from infection of the corresponding pig; only 25% of the positive carcasses were contaminated with the same serotype or genotype found in the corresponding feces or mesenteric lymph nodes. In the other slaughterhouse, 70% of the carcasses were contaminated, and only in 4% was the same genotype or serotype detected as in the feces of the corresponding pigs. The other positive carcasses in both slaughterhouses were contaminated by genotypes present in the feces or lymph nodes of pigs slaughtered earlier that day or from dispersed sources in the environment. In slaughterhouses, complex contamination cycles may be present, resulting in the isolation of many different genotypes circulating in the environment due to the supply of positive animals and in the contamination of carcasses, probably through aerosols.