Flagellar motility mediates biofilm formation in Aeromonas dhakensis.

Aeromonas dhakensis possesses dual flagellar systems for motility under different environments. Flagella-mediated motility is necessary for biofilm formation through an initial attachment of bacteria to the surface, but this has not been elucidated in A. dhakensis. This study investigates the role of polar (flaH, maf1) and lateral (lafB, lafK and lafS) flagellar genes in the biofilm formation of a clinical A. dhakensis strain WT187 isolated from burn wound infection. Five deletion mutants and corresponding complemented strains were constructed using pDM4 and pBAD33 vectors, respectively, and analyzed for motility and biofilm formation using crystal violet staining and real-time impedance-based assays. All mutants were significantly reduced in swimming (p < 0.0001), swarming (p < 0.0001) and biofilm formation using crystal violet assay (p < 0.05). Real-time impedance-based analysis revealed WT187 biofilm was formed between 6 to 21 h, consisting of early (6-10 h), middle (11-18 h), and late (19-21 h) stages. The highest cell index of 0.0746 was recorded at 22-23 h and biofilms began to disperse starting from 24 h. Mutants Δmaf1, ΔlafB, ΔlafK and ΔlafS exhibited reduced cell index values at 6-48 h when compared to WT187 which indicates less biofilm formation. Two complemented strains cmaf1 and clafB exhibited full restoration to wild-type level in swimming, swarming, and biofilm formation using crystal violet assay, hence suggesting that both maf1 and lafB genes are involved in biofilm formation through flagella-mediated motility and surface attachment. Our study shows the role of flagella in A. dhakensis biofilm formation warrants further investigations.

Molecular identification and biofilm-forming ability of Elizabethkingia species.

Recently, Elizabethkingia species have gained attention as a cause of life-threatening infections. The identification via phenotypic methods of three important species- Elizabethkingia meningoseptica, E. anophelis and E. miricola is difficult. Our objectives were to re-assess 30 archived Flavobacterium meningosepticum isolates using 16S rRNA gene sequencing, ERIC-PCR, and biofilm formation assay. Twenty-four isolates were re-identified as E. anophelis and 6 as E. miricola. All of them had the ability to form biofilm as shown in microtiter plate assay based on crystal violet staining. Overall, E. anophelis had a higher specific biofilm formation index compared to E. miricola. A total of 42% (10 out of 24) of E. anophelis were classified as strong, 29% (7 out of 24) as moderate and 29% (7 out of 24) as weak biofilm producers. E. miricola, 17% (1 out of 6) isolates were strong biofilm producers, 50% (3 out of 6) moderate and 33% (2 out of 6) were weak producers. E. anophelis from tracheal secretions were significantly associated with (p = 0.0361) strong biofilm formation. In summary, this study showed that the isolates originally identified as F. meningosepticum were re-classified using the 16S rRNA gene as one of two Elizabethkingia species. The ability of E. anophelis to form strong biofilm in endotracheal tubes indicates their probable role in the pathogenesis of Elizabethkingia infections.

Molecular characterization of putative virulence determinants in Burkholderia pseudomallei.

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis.

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

Aeromonas dhakensis: Clinical Isolates with High Carbapenem Resistance.

Aeromonas dhakensis is ubiquitous in aquatic habitats and can cause life-threatening septicaemia in humans. However, limited data are available on their antimicrobial susceptibility testing (AST) profiles. Hence, we aimed to examine their AST patterns using clinical (n = 94) and non-clinical (n = 23) isolates with dehydrated MicroScan microdilution. Carbapenem resistant isolates were further screened for genes related to carbapenem resistance using molecular assay. The isolates exhibited resistance to imipenem (76.9%), doripenem (62.4%), meropenem (41.9%), trimethoprim/sulfamethoxazole (11.1%), cefotaxime (8.5%), ceftazidime (6%), cefepime (1.7%) and aztreonam (0.9%), whereas all isolates were susceptible to amikacin. Clinical isolates showed significant association with resistance to doripenem, imipenem and meropenem compared to non-clinical isolates. These blacphA were detected in clinical isolates with resistance phenotypes: doripenem (67.2%, 45/67), imipenem (65.9%, 54/82) and meropenem (65.2%, 30/46). Our findings showed that the MicroScan microdilution method is suitable for the detection of carbapenem resistance in both clinical (48.9-87.2%) and non-clinical (4.3-13.0%) isolates. This study revealed that A. dhakensis isolates had relatively high carbapenem resistance, which may lead to potential treatment failure. Continued monitoring of aquatic sources with a larger sample size should be carried out to provide further insights.

Characterization of the relationship between polar and lateral flagellar genes in clinical Aeromonas dhakensis: phenotypic, genetic and biochemical analyses.

Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis.

Serotype distribution and antibiotic susceptibility of group B streptococci in pregnant women.

Group B streptococcus (GBS) is a leading cause of neonatal sepsis and is usually acquired via the woman's birth canal. GBS serotypes isolated from 200 pregnant women were determined. Serotypes V (19%) and VI (17%) were the most frequent followed by serotypes III (12%), Ia (11.5%) and IV (10%); 17% of the strains were non-typable. All isolates were susceptible to penicillin, 96% to erythromycin and 97.5% to clindamycin. The emergence of new GBS serotypes has important implications for vaccine prevention strategies.

Burkholderia thailandensis whole cell antigen cross-reacts with B. pseudomallei antibodies from patients with melioidosis in an immunofluorescent assay.

An immunofluorescent assay (IFAT) using whole cell antigen derived from Burkholderia thailandensis used for detection of total antibodies to Burkholderia pseudomallei, was found to compare favorably with a previous published report on a B. pseudomallei IFAT assay. At a 1:20 cut-off titer, the assay had high sensitivity (98.9%) and satisfactory specificity (92.3%), when tested against sera from 94 patients suspected of melioidosis. Sera from 12 patients with culture proven melioidosis gave absolute concordance with the 2 test antigens. No sera from 50 blood donors had a titer of > or =20. Cross-reactivity with patients' sera positive for Chlamydia, Mycoplasma, Legionella and typhoid was not observed, except for 3 sera from typhus patients and one from a patient with leptospirosis. The major advantage of this assay is that the cultivation and preparation of B. thailandensis as antigen can be carried out in any laboratory with basic microbiological set-up. The serodiagnosis of melioidosis can be made safe for medical laboratory personnel, particularly in B. pseudomallei endemic regions.

Genetic relatedness and novel sequence types of clinical Aeromonas dhakensis from Malaysia.

Aeromonas dhakensis is an emergent human pathogen with medical importance. This study was aimed to determine the sequence types (STs), genetic diversity, and phylogenetic relationships of different clinical sources of 47 A. dhakensis from Malaysia using multilocus sequence typing (MLST), goeBURST, and phylogenetic analyses. The analysis of a concatenated six-gene tree with a nucleotide length of 2994 bp based on six housekeeping genes (gyrB, groL, gltA, metG, ppsA, and recA) and independent analyses of single gene fragments was performed. MLST was able to group 47 A. dhakensis from our collection into 36 STs in which 34 STs are novel STs. The most abundant ST521 consisted of five strains from peritoneal fluid and two strains from stools. Comparison of 62 global A. dhakensis was carried out via goeBURST; 94.4% (34/36) of the identified STs are novel and unique in Malaysia. Two STs (111 and 541) were grouped into clonal complexes among our strains and 32 STs occurred as singletons. Single-gene phylogenetic trees showed varying topologies; groL and rpoD grouped all A. dhakensis into a tight-cluster with bootstrap values of 100% and 99%, respectively. A poor phylogenetic resolution encountered in single-gene analyses was buffered by the multilocus phylogenetic tree that offered high discriminatory power (bootstrap value = 100%) in resolving all A. dhakensis from A. hydrophila and delineating the relationship among other taxa. Genetic diversity analysis showed groL as the most conserved gene and ppsA as the most variable gene. This study revealed novel STs and high genetic diversity among clinical A. dhakensis from Malaysia.

Rapid identification of melioidosis agent by an insulated isothermal PCR on a field-deployable device.

BACKGROUND: Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. METHOD: In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel. RESULTS: All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45-99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. CONCLUSION: This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

Melioidosis in Malaysia.

Melioidosis is an important cause of sepsis in the tropics, is caused by an environmental saprophyte--B. pseudomallei. It affects mainly adults with underlying predisposing condition such as diabetes. The range of symptoms varies from benign and localized abscesses, to severe community-acquired pneumonia to acute fulminating septicaemia with multiple abscesses often leading to death. B. pseudomallei is an intracellular pathogen and some of the virulence mechanisms that govern the complex interaction between the organism and the host have been elucidated. Isolation of B. pseudomallei from bodily fluids of patients remains the "gold standard" in diagnosis but a sensitive and specific serological test can lend support to the diagnosis of melioidosis. Ceftazidime is the treatment of choice for severe melioidosis, but the response is slow. Maintenance or eradication therapy for a prolonged period is necessary to prevent relapse and recurrence. Monitoring IgG antibody levels may be useful as a guideline to determine the duration of eradication therapy.

First Report of Extended-Spectrum ß-Lactamases TEM-116 and OXA-10 in Clinical Isolates of Alcaligenes Species from Kuala Lumpur, Malaysia.

There is an alarming increase in the prevalence of extended-spectrum ß-lactamases (ESBLs) present mainly in Enterobacteriaceae and other nonfermenting gram-negative bacteria, such as Alcaligenes faecalis, which is the only species in that genus that is clinically relevant. We investigated Alcaligenes species from 7 cases (6 inpatients and one outpatient) at our tertiary-care hospital. Four patients had urinary tract infections, and one each had systemic lupus erythematosus, pulmonary stenosis, and diabetic ulcer. All 7 isolates were identified as Alcaligenes spp. based on their 16S rRNA gene sequences, and antibiotic susceptibility was determined using a Vitek 2 system with AST-GN87 cards. All the strains were resistant to cefazolin; 6 were resistant to trimethoprim/sulfamethoxazole; 5 manifested resistance to ampicillin/sulbactam, cefepime, tobramycin, ciprofloxacin, and nitrofurantoin; whereas 5 had multidrug resistance profiles. All the strains (7/7) expressed ESBL activity; PCR screening and sequencing showed evidence of genes blaTEM-116 (7/7) and blaOXA-10 (4/7), and we believe that this is the first report on the presence of TEM-116 and OXA-10 in an Alcaligenes spp. A combination of the 2 genes was present in 4 strains. All 7 strains were found to harbor at least one ESBL gene probably contributing to the drug resistance.

Comparison by electron microscopy of intracellular events and survival of Burkholderia pseudomallei in monocytes from normal subjects and patients with melioidosis.

INTRODUCTION: Burkholderia pseudomallei (B. pseudomallei) has been shown to persist intracellularly in patients with melioidosis, until reactivated by decreasing immunocompetence. We have previously demonstrated by transmission electron microscopy, the internalisation of B. pseudomallei by human macrophages and the occurrence of phagosome-lysosome fusion. METHODS: Phagocytosis and electron microscopy were used to compare the rate of phagosome-lysosome fusion and the intracellular survival of B. pseudomallei using monocytes obtained from five patients with melioidosis and five normal healthy adults. RESULTS: Ingested bacilli were seen in various stages of degradation, with a few remaining viable within phagolysosomes, and the proliferation of these viable bacteria was observed. Phagocytosis of B. pseudomallei by normal macrophages was two-fold higher than uptake by the melioidosis macrophages (p-value is less than 0.001). Three times more phagolysosomes were present in the normal macrophages, indicating that fusion occurred slowly and inefficiently in the melioidosis macrophages (p-value is less than 0.001), resulting in higher number of organisms within the melioidosis macrophages (p-value is less than 0.001). Both variables were inversely related to each other. CONCLUSION: Our observations suggest that phagolysosome fusion occurred slowly and inefficiently in monocytes of patients with melioidosis, leading to an increased number of intracellular organisms compared to monocytes obtained from healthy donors.

Rhodococcus equi--an emerging human pathogen in immunocompromized hosts: a report of four cases from Malaysia.

Rhodococcus equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized hosts. We describe four immunocompromized patients who had serious R. equi infections with an overall mortality of 75%. The natural habitat of R. equi is soil, particularly soil contaminated with animal manure. Necrotizing pneumonia is the commonest form of infection but extrapulmonary infections, such as wound infections and subcutaneous abscess, have also been described in humans. R. equi is cultured easily in ordinary non-selective media. Large, smooth, irregular colonies appear within 48 hours. It is a facultative, intracellular, nonmotile, non-spore forming, gram-positive coccobacillus, which is weakly acid-fast staining and bears a similarity to diphtheroids. It forms a salmon-colored pigment usually after 48 hours incubation. A particular characteristic of this organism is that it undergoes synergistic hemolysis with some bacteria on sheep blood agar. R. equi may be misidentified as diphtheroids, Mycobacterium species, or Nocardia. In vitro R. equi is usually susceptible to erythromycin, ciprofloxacin, vancomycin, aminoglycosides, rifampin, imipenem and meropenem. The organism can be difficult to eradicate, making treatment challenging. Increased awareness of the infection may help with early diagnosis and timely treatment.

Burkholderia pseudomallei lectins: occurrence and expression.

Lectins, also known as sugar binding proteins, play an essential role in the initiation of bacterial infections and biofilm production. To date, several lectins of Gram-negative bacteria such as Pseudomonas aeruginosa, Burkholderia cenocepacia, Ralstonia solanacearum and Chromobacterium violaceum have been identified. There are no published reports on the presence of lectins in Burkholderia pseudomallei, the causative agent of melioidosis. The aim of this study was to identify possible lectin genes of B. pseudomallei and generate recombinant proteins for assessment of hemagglutinating activity. Seven hypothetical lectins of B. pseudomallei were retrieved from the UniProt database. Four lectin domains, i.e., ricin B, C-type, H-type and Bulb-type lectins were identified. In silico analysis using a ligand binding site prediction server (3DLigandSite) predicted the presence of Nacetylglucosamine and calcium binding sites in two C-type lectins. Four recombinant proteins with the molecular weights of 11.7, 30.2, 36.2 and 46.4 kDa were expressed from the cloned genes; however none of them expressed any hemagglutinating activity. Further characterization of B. pseudomallei lectins may be able to provide insights into bacterial-host interaction that are required to initiate infections.

Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay.

BACKGROUND: In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay. METHODS: A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997-2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth. RESULTS: The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation. CONCLUSIONS: The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.

Focal non-typhoidal Salmonella infections from a single center in Malaysia.

A retrospective review of patients with focal non-typhoidal Salmonella (NTS) infection was performed to determine its features and outcome. All patients with focal NTS infection admitted to the University of Malaya Medical Center, Malaysia, from 1993 to 2002 were studied. More than half (58%) of the 35 cases (54% male, median age 39 years, range 1.5 months to 79 years) were immunocompromized or had chronic medical conditions. One-third of the patients (34%) had superficial infections (lymphadenitis or subcutaneous tissue infection) and all recovered with antimicrobial therapy alone. Deep infections (66%) noted were: meningitis (9%), osteomyelitis or arthritis (26%), abscesses of the gastrointestinal tract or adjacent organs (20%), and others (11%). Deep infections were more likely to occur in the extremes of age (<6 months or >60 years, p< 0.04), associated with adverse outcomes with an overall mortality rate of 9%, or required major surgery (15%).

An electronmicroscopic study of the interaction of Burkholderia pseudomallei and human macrophages.

B. pseudomallei has been shown to persist intracellularly in melioidosis patients until reactivated by decreasing immunocompetence. We have shown by transmission electron microscopy the internalization of B. pseudomallei by human macrophages via conventional phagocytosis enclosed within membrane-bound vacuoles or phagosomes. Ferritin labeled lysosomes provided evidence of phagosome-lysosome fusion. Ingested bacilli were designated as "intact" or "damaged" on the basis of their ultrastructural features. An intact bacterium was seen with low electron opaque central nuclear region surrounded by dense bacterial cytoplasm, bounded externally by bacterial plasma membrane and cell wall. In contrast, B. pseudomallei were considered damaged when seen with cavitation within the central nuclear region, separation of bacterial cytoplasm from the cell wall, herniation of cytoplasmic contents and lamination of bacterial cell wall and its surrounding electron transparent zone. Our observations indicate that the microbicidal mechanism(s) in B. pseudomallei-infected macrophages failed to ensure complete clearance of the organism and this failure probably facilitates intracellular persistence and proliferation, and this may be one of the survival strategies adopted by this organism.

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia.

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.

Genome size variation among recent human isolates of Salmonella typhi.

We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3') and SpeI (5'-ACTAGT-3'), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.