Monoclonal antibodies delineate multiple epitopes on the O antigens of Salmonella typhi lipopolysaccharide.

Fifteen monoclonal antibodies (MAbs) directed against Salmonella typhi were produced and characterized. The specificities of the antibodies were determined by their binding patterns in an enzyme immunoassay, with a panel of lipopolysaccharides isolated from different bacteria. Seven MAbs reacted with S. typhi, Salmonella enteritidis, and Salmonella dublin (all belonging to serogroup D). One MAb also reacted with Salmonella paratyphi A and S. paratyphi B. Five MAbs reacted with S. typhi, S. enteritidis, S. dublin, and S. paratyphi B. Two MAbs did not bind to any lipopolysaccharide but showed reactivity with bacterial sonic extracts isolated from S. typhi, S. paratyphi A, S. paratyphi B, Escherichia coli, and Shigella sonnei. These antibodies would be helpful in studying the complexity of antigenic determinants expressed by S. typhi and the nature of the antibody response during typhoid and paratyphoid fevers and also in the diagnosis of the disease.     

Present phage types and antibiotic susceptibility of salmonellae.

A total of 168 strains of Salmonella were isolated in the Command Pathology Laboratory (WC) Delhi Cantt during the year 1990. Out of this, 143 were Salmonella typhi, 17 Salmonella paratyphi A, 7 Salmonella typhimurium and 1 Salmonella manhattan. The commonest phage type and biotype of Salmonella typhi was type E1 and type 1 respectively. The dominant biotype of Salmonella paratyphi A was type I. There was a very high degree of multidrug resistance of most of the strains. But all the strains were sensitive to ciprofloxacin and norfloxacin.     

Monoclonal antibodies against flagellar antigen of Salmonella typhi

Two hybrid cell clones secreting monoclonal antibodies against flagellar antigen isolated from Salmonella typhi, were produced and characterized. The antibodies bound specifically to the flagellar strain of S. typhi and did not show any reactivity with a flagellar S. typhi or with flagellar strains of S. dublin, S. paratyphi A, S. paratyphi B, S. typhimurium, E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The antibodies recognized a determinant present on a group of proteins migrating between 45 Kd and 60 Kd. These monoclonal antibodies would be useful reagents for clinical and epidemiological studies.     

Growth of Typhoid and Paratyphoid Bacilli in Intravenously Infected Mice

The in vivo growth of Salmonella paratyphi A, S. paratyphi B, S. paratyphi C, and S. typhi, as well as of an S. typhi-typhimurium hybrid, was studied in three different strains of mice. S. paratyphi A and B and S. typhi demonstrated very little growth potential in any of the intravenously infected mice, even after as many as 20 serial mouse passages. It was noted, however, that small numbers of viable S. paratyphi B and S. typhi persisted in the spleens of infected mice for up to 28 days. Salmonella paratyphi C and the S. typhi-typhimurium hybrid gave rise to progressive systemic infections beginning from very small intravenous inocula. The median lethal doses for the C57B1 strain of mouse were about five organisms. The relevance of these findings with regard to the development of an animal model for studying human typhoid fever vaccines is discussed.     

Molecular analysis of ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates

Objective: To study the mechanisms of antibiotic resistance in Salmonella typhi and Salmonella paratyphi B clinical isolates, and the clonality of resistant strains.
Method: Antibiotic susceptibility was tested by disk-agar diffusion. Conjugation experiments and plasmid analysis by agarose gel electrophoresis after Eco RI digestion were followed by hybridization to a digoxigenin-labeled TEM-type β-lactamase probe. DNA fingerprints were obtained by pulsed-field gel electrophoresis of Xba I-digested chromosomal DNA.
Results: Three S. typhi isolates (7% of the isolates studied), of which one was ampicillin resistant and the other two multiresistant (ampicillin, chloramphenicol, tetracycline, sulfamethoxazole/trimethoprim and streptomycin), and two ampicillin-resistant S. paratyphi B isolates (25% of the isolates studied) were further evaluated. A 34-MDa conjugative plasmid, previously isolated from Salmonella enteritidis , conferred ampicillin resistance. A 100-MDa conjugative plasmid encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim, as well as ampicillin. Chromosomal fingerprinting revealed two distinct resistant strains for each serovar which were different from a matched set of sensitive S. typhi strains.
Conclusions: Two conjugative, TEM-type β-lactamase-encoding plasmids conferred ampicillin resistance to S. typhi and S. paratyphi B. The 34-MDa plasmid was identical to that previously characterized from S. enteritidis , while the 100-MDa plasmid also encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim. Resistant isolates did not belong to a single clone but rather represented distinct strains.     

Cross-reactions in cell-mediated immunity to Salmonella causing enteric fever

Cross-reactivity in a delayed-type hypersensitivity (DH) response was studied in mice immunised with live Salmonella typhi, S. paratyphi A and S. paratyphi B. Extensive cross-reactions outside the serogroup limits were observed. The ability of DH cross-reacting and non-cross-reacting sonicates to generate activated macrophages was studied in mice immunised 3 months earlier with S. paratyphi B. Whereas DH cross-reacting S. poona sonicate generated activated macrophages the non-cross-reacting S. typhi sonicate did not. To determine whether infections due to diarrhoea-causing salmonellae generated cross-reactive cell-mediated immune responses against enteric fever-causing organisms, similar reverse experiments were performed in mice immunised with S. enteritidis. S. paratyphi A sonicate generated both effector responses, i.e., DH and activated macrophages.     

Characterization of the Salmonella paratyphi C Vi polysaccharide.

The Vi capsular polysaccharide (Vi) is both a virulence factor and a protective antigen of Salmonella typhi; its pathogenic role for Salmonella paratyphi C is less well understood. We found no differences between the antigenic and immunogenic properties and the structure of the Vi from representative strains of S. paratyphi C, S. typhi, and Citrobacter freundii. There were, however, differences in both the amount produced per cell and the degree of association with the cell among the Vi from the three species of Enterobacteriaceae. S. paratyphi C produced less Vi than both the wild-type S. typhi and C. freundii did, and it showed the fastest release of Vi into the media. These findings may provide an explanation for the inability of the Vi to inhibit completely the agglutination of S. paratyphi C by anti-O sera. In an outbreak of enteric fever caused by S. paratyphi C, 66 of 78 isolates (85%) were Vi positive.     

Monoclonal antibodies against Salmonella porins: generation and characterization.

Monoclonal antibodies (mAbs) were generated against porins, one of the major outer membrane proteins of Salmonella typhi. Six clones, designated MP1, MP2, MP3 (IgG2ak), MPN4, MPN6 (IgG1k) and MPN5 (IgG2bk) were characterized by enzyme immunoassay (ELISA) for their reactivity to porins from S. typhi, Salmonella paratyphi A, S. paratyphi B, S. paratyphi C, Salmonella choleraesuis, Salmonella enteritidis, Salmonella krefeld, Salmonella panama, Salmonella typhimurium, Escherichia coli B, Shigella flexneri 1b and Pseudomonas aeruginosa. All the clones positive for S. typhi porins showed varying reactivity towards several Salmonella species. However, none of them was positive for porins from other Gram-negative bacteria or for lipopolysaccharide (LPS). The affinity constant of these mAbs, except MPN4, was found to be in the higher range. Dot ELISA revealed that the mAbs recognized porins only in their native form. The results of inhibition ELISA using horseradish peroxidase (HRP)-conjugated MP1 suggest that the clones MP1, MP2, MP3, MPN5 and MPN6 secreted antibodies to identical epitope(s) of a 36-kDa peptide and MPN4 to a different epitope of a 35-kDa peptide. The possible applications of these mAbs were discussed.     

Multidrug resistant Salmonella

A total of 326 salmonella strains was isolated and studied from suspected enteric fever cases in Mumbai (Bombay) during a 2 year period from May 1992 to July 1994. These were identified using standard biochemical and serological tests. Bacteriophage typing, antibiotic sensitivity and conjugation experiments were also carried out. S. typhi was the most common serotype accounting for 75.46% of the strains. Among S. typhi strains 87% were biotype I and 13% were biotype II. 9.5% strains were of S. paratyphi A, 5.52% of S. typhimurium, 4.60% of S. worthington, 4.30% of S. havana and 0.62% of S. enteritidis. The commonest bacteriophage type of S. typhi was E1, and of S. paratyphi A type 1, whereas 88.88% strains of S. typhimurium were untypable. Most of the strains were multidrug resistant including commonly used antibiotics such as chloramphemicol, ampicillin, and cotrimaxazole. Quinolone derivatives such as Ciprofloxacin were found to be the most effective drugs. In the conjugation experiments there was direct transfer of resistance pattern and enbloc transfer of resistance was observed in most strains. Salmonella typhi is still the most commonly encountered species. There is an alarming increase in multidrug resistance.     

Development of nested PCR based on the ViaB sequence to detect Salmonella typhi.

For a rapid diagnosis of typhoid fever, we developed a nested PCR based on the nucleotide sequence encoding the Vi antigen. All Salmonella typhi strains along with a Salmonella paratyphi C strain were PCR positive. This assay was able to detect S. typhi at the single-cell level.     

Salmonella types and antibiotic susceptibility: a six months survey

A total of 51 Salmonella strains were isolated during the six month period of May 1998 till October 1998. Of these, 41 (80.3%) were Salmonella typhi, 5 (9.8%) Salmonella pararyphi A,2 (3.9%) Salmonella worthington and 1 (1.9%) Salmonella senftenberg. The prevalent phage and biotype of Salmonella typhi was E1 (75%) and type 1 (90.2%) respectively. The commonest pattern of multiple drug resistance in Salmonella typhi was ACCoT and 92.5% of these belonged to phage E1. Out of the five Salmonella paratyphi A, one belonged to phage type 1 and the others were untypable. Similarly both the strains of Salmonella typhimurium were untypable. Thus the predominant isolate was Salmonella typhi and the commonest phage and biotype were E1 and biotype1 respectively.     

Electrophoretic analysis of Salmonella typhi and other bacteria

A comparative study involving SDS-PAGE of Salmonella typhi and other Bacteria was conducted. Protoplasmic antigens of Salmonella typhi. Salmonella paratyphi A, Salmonella typhimurium, Proteus sp, Klebsiellas sp. Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus were separated and compared on SDS-PAGE followed by checking of their cross reactivity by gel diffusion using antisera raised against whole cell and lysates of Salmonella typhi. Lines of identity between Salmonella typhi, Salmonella paratyphi A and Salmonella typhimurium were observed. No lines of identity were seen among Salmonella typhi, Pseudomonas aeruginosa and Sfaphylococcus aureus.     

伤寒杆菌鞭毛抗原酶联免疫分析的建立及初步应用

作者利用纯化的伤寒杆菌鞭毛抗原，制备出抗伤寒直菌鞭毛抗原单克隆抗体。实验发现，四株单抗体与伤塞杆菌鞭毛抗原和伤寒杆菌发生免疫反应，与甲、乙、丙副伤寒杆菌、大肠杆菌及部分沙门氏菌无交叉反应。选用其中２株单抗分别作为包被抗体和标记抗体，建立检测伤寒杆菌鞭毛抗原的夹心ＥＬＩＳＡ。３０名正常人和４２例非伤寒发热待查患者血清标本经验测为阴性，１２例途塞患者血清标本为阳性。本文方法可直接用血清进行检测，具有早     

Salmonellosis in Goa.

During the period 1982-86, a total of 657 Salmonella strains were isolated from various clinical samples processed in the Microbiology laboratory of Goa Medical College, Bambolim, Goa. The strains were distributed amongst 23 different Salmonella serotypes. The commonest serotypes encountered were S.typhimurium (66%) and S.typhi (24%), the other serotypes were S.bareilly (5.4%), S.paratyphi B (1.2%), S.newport (1.2%) and S.chester (0.8%). Stool samples yielded the maximum Salmonella isolates of which the S.typhimurium was the highest followed by S.bareilly.     

Semisolid selective-motility enrichment medium for isolation of salmonellae from fecal specimens.

A semisolid selective-motility enrichment medium for the isolation of salmonellae from fecal specimens was developed which was based on Rappaport enrichment broth. During a 7-year period more than 30,000 stool samples were tested. The medium showed a high specificity (95.1%) and sensitivity (80.3%) when compared with MacConkey agar, SS agar, and brilliant green agar (after Selenite-F Enrichment [BBL Microbiology Systems]). Furthermore, our isolation rate of Salmonella species from fecal samples showed an increase of 22.3% when this semisolid medium was added to the routine culture media. Growth could easily be interpreted. The medium has a bias toward the isolation of Salmonella paratyphi B, but it is unsatisfactory for detecting the nonmotile strains Salmonella typhi and S. paratyphi A.     

Changing trends in the antibiograms of Salmonella isolates at a tertiary care hospital in Hyderabad

The present study was undertaken to compare the changing trends of antibiograms of Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A isolates. A total of 80 isolates of salmonella obtained from blood cultures between 2001-2004 were included in the study. Identification and antibiotic sensitivities of the isolates were performed by using mini API (bio Merieux, France). Sixty isolates were identified as Salmonella enterica serovar Typhi and 20 were identified as Salmonella enterica serovar Paratyphi A. More than 67% of S.typhi and 80% of S.paratyphi A isolates were sensitive to chloramphenicol. Sensitivity of S.typhi isolates to cephalosporins was found to have increased from 2001-2004 while that of S.paratyphi A showed a decline. With increasing resistance to ciprofloxacin and the possibility of re-emergence of sensitivity to chloramphenicol, the policy of empirical treatment of enteric fever needs to be rationalized.     

Characterization of monoclonal antibodies to protein antigen of Salmonella typhi

Two monoclonal antibodies were produced against protein antigens of Salmonella typhi. One of the antibodies (STP14) belongs to the immunoglobulin G1K subclass, and the other (STP13) was assigned to the immunoglobulin G2a(kappa) subclass. Both antibodies could recognize the 34.0-kilodalton protein antigen from S. typhi. The specificity of these antibodies was tested by immunoblotting with a panel of crude protein antigens from 12 bacteria causing enteric fever and enteric fever-like illness: S. typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. choleraesuis, S. enteritidis, S. krefeld, S. panama, S. typhimurium, Escherichia coli, Pseudomonas pseudomallei, and Yersinia enterocolitica. In a modified double-antibody sandwich enzyme-linked immunosorbent assay they could detect the protein antigen at ca. 0.6 microgram/ml. These monoclonal antibodies should be of great value in the diagnostic test for detecting S. typhi antigen in samples of bodily fluids isolated from patients with typhoid fever and in studies of the chemical structure and other immunological properties of this 34.0-kilodalton protein.     

Use of Rambach Propylene Glycol Containing Agar for identification of Salmonella spp.

When grown on Rambach Propylene Glycol Containing Agar (Rambach agar), 216 of 230 (93.9%) Salmonella organisms isolated from patients and 54 of 62 (87.1%) Salmonella stock cultures produced a crimson-colored growth. Of the 14 clinical Salmonella isolates which displayed colors other than crimson, 8 were Salmonella typhi, 2 were Salmonella paratyphi A, and 4 belonged to other commonly isolated serotypes. All eight Salmonella stock cultures which failed to produce a crimson color belonged to rarely isolated serotypes. In contrast, of 83 non-Salmonella stock cultures distributed among 29 bacterial species, none produced a crimson color. These results suggest that while Rambach agar cannot preidentify S. typhi and S. paratyphi A, the medium can be used for the presumptive identification and can assist in the definitive identification of the overwhelming majority of Salmonella isolates.     

甲型副伤寒杆菌nmpC基因原核表达及表达产物免疫保护作用

目的 构建甲型副伤寒杆菌外膜蛋白基因nmpC的原核表达系统,确定其重组表达产物rNmpC免疫原性和保护作用,了解甲型副伤寒杆菌临床菌株nmpC基因携带及表达率.方法 采用PCR和T-A克隆法从甲型副伤寒杆菌临床株JH01中获得nmpC基因克隆并构建其原核表达系统.采用SDS-PAGE和Bio-Rad凝胶图像分析系统检测rNmpC表达情况及其产量,采用免疫扩散法、Western blot和微量肥达试验鉴定其抗原性和免疫应答性.采用PCR和ELISA分别检测98株甲型副伤寒杆菌临床菌株nmpC基因携带及表达率.采用小鼠感染模型了解rNmpC对甲型副伤寒杆菌致死性感染的免疫保护作用.结果 与报道的相关序列比较,所克隆的nmpC基因核苷酸和氨基酸序列相似性均为100%.rNmpC表达量约为细菌总蛋白的30%.rNmpC免疫家兔可产生抗体并能与甲型副伤寒杆菌全菌抗血清产生阳性Western杂交信号.所有甲型副伤寒杆菌菌株均携带nmpC基因并表达NmpC蛋白,但伤寒杆菌、乙型及丙型副伤寒杆菌未检出nmpC基因.100μg和200μgrNmpC对感染小鼠的免疫保护率分别为41.7%(5/12)和66.7%(8/12).rNmpC免疫小鼠或保护试验存活小鼠血清仅对甲型副伤寒杆菌H抗原产生1∶5～1∶40的凝集效价.结论 NmpC是甲型副伤寒杆菌独有的序列保守、分布广泛且自然表达的外膜蛋白抗原,该外膜蛋白具有良好的免疫原性和一定的免疫保护作用,可作为多价甲型副伤寒杆菌基因工程疫苗候选抗原.     

Monoclonal antibodies against two discrete determinants on Vi capsular polysaccharide.

Vi is a linear homopolymer of 1,4 N-acetyl galactosaminuronic acid. It is present in S. typhi and some other members of Enterobacteriaceae. Vi antigen of S. typhi has been associated with the virulence of the organism and a vaccine based upon this antigen has been found to confer immunity against typhoid. In this paper, we report production and characterization of four hybrid cell clones secreting monoclonal antibodies against Vi capsular polysaccharide. Binding analysis using different derivatives of Vi showed that three monoclonal antibodies reacted with the antigenic determinant constituted by O-acetyl group and one recognised the epitope constituted by N-acetyl and carboxyl groups together. All the antibodies bound to Vi positive strains of S. typhi and did not show any significant reactivity with Vi negative strains of S. typhi, S. paratyphi A, S. paratyphi B and E. coli. Besides their utility in studying the sub-specificity of antibodies produced after vaccination with Vi, these antibodies would be helpful in the diagnosis of typhoid fever.