ST Isolates Research Articles

Colonizing and Virulence Factors in Enterotoxigenic Escherichia coli from Peru.

Enterotoxigenic Escherichia coli (ETEC) ranks among the most relevant diarrheagenic pathogens. Efforts to design vaccines to fight ETEC have been focused on colonizing factors (CFs) and atypical virulence factors (AVF). An effective vaccine must account for differences in the regional prevalence of these CFs and AVFs to be truly effective in a given area. In the present study, the presence of 16 CFs and 9 AVFs, as well as the heat-stable (ST) variants (STh or STp), was established by polymerase chain reaction in 205 Peruvian ETEC isolates (120 from diarrhea cases and 85 from healthy controls). Ninety-nine (48.3%) isolates were heat-labile, 63 (30.7%) ST, and 43 (21.0%) presented both toxins. Of ST isolates, 59 (28.8%) possessed STh, 30 (14.6%) STp, five (2.4%) both STh and STp, and 12 (5.8%) were not amplified for any variant tested. The presence of CFs was associated with diarrhea (P < 0.0001). The presence of eatA as well as concomitant presence of CSI, CS3, and CS21 and of C5 and C6 was statistically related to diarrhea cases. The present results suggests that, if effective, a vaccine considering CS6, CS20, and CS21, together with EtpA, would provide protection against 64.4% of the isolates analyzed, whereas the addition of CS12 and EAST1 would lead to 83.9% coverage. Large studies are needed to establish both the ideal candidates to be considered to develop a vaccine effective in the area, and continuous surveillance is needed to detect displacement of circulating isolates that may compromise future vaccines.

ANTIBIOTIC RESISTANCE PATTERN IN BACTERIA ISOLATED FROM ORTHOPAEDIC RELATED INFECTIONS

Background. Orthopaedics &amp; Implant related infections continue to pose a problem for the orthopaedicians. An increase in the number of multidrug resistant bacteria stresses the value of an adequate diagnosis, leading to a proper therapy of these patients. To isolate bacteria causing orthopaedic related infecti Objective. Materials ons and determine their antibiotic resistance pattern. &amp; methods. A total of 111 samples were received during the study period in the Department of Microbiology &amp; were processed. Bacteria causing orthopaedic related infections were isolated and their antimicrobial susceptibility testing was determined. . There were 66 (59%) isolates Results that showed culture positivity &amp; were processed. Among which, gram positive cocci were 35 (53%) &amp; gram negative bacilli were 31 (47%). The common gram positive cocci were Methicillin sensitive Staphylococcus aureus 31.81% followed by CoNS 10.6% &amp; MRSA 10.6% While the common gram negative bacilli were Pseudomonas aeruginosa 18.18%, Klebsiella pneumoniae 12.12%, Escherichia coli 7.5%, Citrobacter freundii 4.5%, Proteus mirabilis 3.03% &amp; Acinetobacter baumannii 1.51%. Most of the gram positive cocci were resistance to the following antibiotics- ciprooxacin, penicillin, erythromycin. Most of the gram negative bacilli were resistance to the cephalosporins group of drugs mainly ceftriaxone &amp; ceftazidime . Gram positive cocci were the commone Conclusion st isolates from orthopaedic related infection as compare to gram negative bacteria. Antibiotic resistance pattern should be given adequate stress so as to get better results regarding orthopaedic related infections

Persistence of Listeria monocytogenes ST5 in Ready-to-Eat Food Processing Environment.

Most human listeriosis is foodborne, and ready-to-eat (RET) foods contaminated by Listeria monocytogenes during processing are found to be common vehicles. In this study, a total of four L. monocytogens STs (ST5, ST121, ST120, and ST2) have been identified in two RTE food plants from 2019 to 2020 in Shanghai, China. The L. monocytogenes ST5 was predominant in one RTE food processing plant, and it persists in the RTE meat processing plant with continued clone transmission. The genetic features of the four STs isolates were different. ST5 and ST121 had the three genes clpL, mdrL, and lde; however, ST120 and ST2 had two genes except for clpL. SSI-1was present in ST5, ST121, and ST120. Additionally, SSI-2 was present only in the ST121 isolates. ST120 had all six biofilm-forming associated genes (actA, prfA, lmo0673, recO, lmo2504 and luxS). The ST2 isolate had only three biofilm-forming associated genes, which were prfA, lmo0673, and recO. The four ST isolates had different biofilm formation abilities at different stages. The biofilm formation ability of ST120 was significantly higher when grown for one day. However, the biofilm formation ability of ST120 reduced significantly after growing for four days. In contrast, the biofilm formation ability of ST5 and ST121 increased significantly. These results suggested that ST5 and ST121 had stronger ability to adapt to stressful environments. Biofilms formed by all four STs grown over four days can be sanitized entirely by a disinfectant concentration of 500 mg/L. Additionally, only ST5 and ST121 biofilm cells survived in sub-lethal concentrations of chlorine-containing disinfectant. These results suggested that ST5 and ST121 were more resistant to chlorine-containing disinfectants. These results indicated that the biofilm formation ability of L. monocytogenes isolates changed at different stages. Additionally, the persistence in food processing environments might be verified by the biofilm formation, stress resistance, etc. Alternatively, these results underlined that disinfectants should be used at lethal concentrations. More attention should be paid to ST5 and ST121, and stronger surveillance should be taken to prevent and control the clonal spread of L. monocytogenes isolates in food processing plants in Shanghai.

Changes in antimicrobial resistance and molecular attributes of Salmonellae causing enteric fever in Kolkata, India, 2014–2018

Globally, enteric fever caused by Salmonella Typhi (S. Typhi, ST) and S. Paratyphi A (SPA) remain one of the major diseases of public health importance. In this study, a total of 457 (380 ST, 77 SPA) blood isolates were collected from three tertiary care hospitals in Kolkata during 2014–18. Additionally, 66 (3.4%) ST and 5 (0.25%) SPA were recovered from blood culture of 1962 patients attending OPD of one pediatric hospital during 2016–18. The study isolates were tested for antimicrobial resistance (AMR) profiles; AMR genes; molecular sub-types by PFGE, MLVA and CRISPR. Among the total 446 ST and 82 SPA isolates, fluoroquinolone (FQ) resistance was very common in both serovars. Ciprofloxacin resistance of 24.9% and 9.8% & ofloxacin resistance of 20.9% and 87.8% were found in ST and SPA respectively. Majority (>70%) of the isolates showed decreased susceptibility to ciprofloxacin (DCS). A single point mutation in gyrA gene (S83F) was responsible for causing DCS in 37.5% (n = 42/112) ST and 63% (n = 46/73) SPA isolates. Multidrug resistance (MDR) was found only in 3.4% ST isolates and encoded the genes blaTEM-1, catA, sul, strA-strB, class 1 integron with dfrA7. All MDR ST (n = 15) possessed non-conjugative non-IncHI1 (180 kb) plasmid except one having conjugative IncHI1 (230 kb) plasmid and one without plasmid. The MDR genes were integrated near chromosomal cyaA gene site in ST with/without the presence of plasmid (nonIncH1). Almost 65.7% resistant ST belonged to H58 haplotype. PFGE showed clonally related isolates with 81% similarity in ST and 87% in SPA. Similarly, CRISPR typing showed less diversity among the isolates. However, the isolates (ST and SPA) were found to be more diverse by MLVA typing (D value 0.987 and 0.938). The study reports decrease in MDR and increase in FQ resistance among typhoidal Salmonella isolates over the years giving interesting information for enteric fever treatment.

Pathogenic Salmonella weakens avian enriched blood monocytes through ATP depletion, apoptosis induction and phagocytosis inefficiency

Salmonella enterica Subsp enterica serovar Typhimurium (S. Typhimurium, ST) is one of the most important serovars of the genus Salmonella in human and animals. Because of its intracellular tropism, monocytes/macrophages are pivotal in killing of Salmonella serovars; they are also responsible for transporting of ST to extra-intestinal organs. To investigate the effect of the ST on the functions of avian innate immune cells, almost homogeneous enriched monocytes (EMo) were isolated from peripheral blood mononuclear cells of 2–3 weeks-old of healthy broilers. The EMo were then divided in three groups: control (media only), treatments (challenged with ST clinical isolates) and [doxorubicin (Dox), specifically as positive control for EMo apoptosis] groups. Cellular-molecular damage caused by ST in EMo was assessed with bioluminescence (for caspase-3, 7, and 9 activities and intracellular ATP content), chemiluminescence (for pro/anti-oxidant capacities) and flow cytometry (for apoptosis/necrosis). Further, phagocytosis capacity of post-ST challenged EMo was assessed using a flow cytometry-based internalisation of FITC-loaded polystyrene microparticles. Like the effects of Dox, in post-ST challenged EMo much higher caspase-3, 7 and 9 activities and ATP depletion along with decreased phagocytosis capacity and anti-oxidant load were observed. The results herein indicate that ST weakens EMo particularly through caspases activation/apoptosis. These findings can open a new window on the molecular aspects of Salmonella-macrophage interactions and immunopathology/pathogenicity of salmonellosis in animals especially avian species.

649. The Clinical Significance of Sequence Type 17 of Vancomycin-Resistant Enterococcus faecium

BackgroundSequence type (ST) 17 of vancomycin-resistant Enterococcus faecium (VREF) is known to be associated with nosocomial isolates. However, there is no evidence of the effect of ST17 VREF on the patient`s clinical outcome. We conducted a retrospective cohort study to identify ST17 VREF would contribute to developing subsequent bacteremia among VREF-colonized patients.MethodsVREF-colonized patients and its non-repetitive rectal VREF isolates were collected between March 2014 and February 2015. Subsequent bacteremia event within 1 year after colonization was reviewed from electronic medical records. STs were identified by multi-locus sequence typing. Cohort was defined as VREF with ST17 or non-ST17. Multivariable cox regression model was used to adjust effect of ST17 for developing subsequent bacteremia. If available, pulsed field gel electrophoresis (PFGE) was conducted to compare similarity between rectal and blood VREF isolates.ResultsFifty-two patients with ST17 and 169 patients with non-ST17 VREF carriage were included in each cohort. There were six cases and 10 cases of subsequent bacteremia in cohorts ST17 and non-ST17, and 1-year VREF bacteremia free rates were 85.9% and 90.2%, respectively. There was no significant difference of subsequent bacteremia (P = 0.257) in log-rank test. However, after adjusted in multivariable models, VREF ST 17 was associated with subsequent bacteremia (adjusted relative risk, 4.02; 95% CI, 1.32–12.29, P = 0.015). Of 16 patients who had developed to subsequent VREF bacteremia, 12 VREF blood isolates could be analyzed. Only six cases (50%) of rectal and blood isolates had identical ST, whereas all available ST17 VREF cases (four cases) had identical ST and PFGE pattern (Figures 1 and 2). Patients who had identical ST isolates had shorter time difference than those who had non-identical ST isolates (P = 0.041).ConclusionIn our study, ST17 VREF was risk factors of subsequent bacteremia and the strain that showed strong concordance between rectal and blood isolates. Further study is needed to improve clinical outcome of patients carrying VREF using genotype data of rectal VREF isolates.Figure 1:Figure 2:Disclosures All authors: No reported disclosures.

Population structure of Environmental and Clinical Legionella pneumophila isolates in Catalonia

Legionella is the causative agent of Legionnaires’ disease (LD). In Spain, Catalonia is the region with the highest incidence of LD cases. The characterisation of clinical and environmental isolates using molecular epidemiology techniques provides epidemiological data for a specific geographic region and makes it possible to carry out phylogenetic and population-based analyses. The aim of this study was to describe and compare environmental and clinical isolates of Legionella pneumophila in Catalonia using sequence-based typing and monoclonal antibody subgrouping. A total of 528 isolates were characterised. For data analysis, the isolates were filtered to reduce redundancies, and 266 isolates (109 clinical and 157 environmental) were finally included. Thirty-two per cent of the clinical isolates were ST23, ST37 and ST1 while 40% of the environmental isolates were ST284 and ST1. Although the index of diversity was higher in clinical than in environmental ST isolates, we observed that clinical STs were similar to those recorded in other regions but that environmental STs were more confined to particular study areas. This observation supports the idea that only certain STs trigger cases or outbreaks in humans. Therefore, comparison of the genomes of clinical and environmental isolates could provide important information about the traits that favour infection or environmental persistence.

Emergence of multidrug-resistant sequence type 45 strains among mecA-positive borderline oxacillin-resistant Staphylococcus aureus causing bacteraemia in a medical centre in Taiwan

Clinical data related to bacteraemia caused by mecA-positive borderline oxacillin-resistant Staphylococcus aureus (BORSA) isolates [minimum inhibitory concentrations (MICs) of 0.5–4 mg/L] are limited. Patients aged ≥15 years with bacteraemia due to BORSA who were admitted to the emergency department of a Taiwanese tertiary hospital between January 2001 and December 2015 were evaluated. Clinical characteristics of patients with bacteraemia caused by BORSA, methicillin-susceptible S. aureus and methicillin-resistant S. aureus (MRSA) were compared. MICs of 12 antibiotics were determined by agar dilution. Genetic characteristics of 64 available BORSA isolates were analysed by detection of the SCCmec gene, the mecA promoter region sequence and multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) was performed to identify the clonality of the emerging ST isolates. The quinolone resistance genes were analysed for the emerging ciprofloxacin-resistant ST45 clone isolates. Among the 65 BORSA bacteraemic patients, in-hospital mortality was 24.6%, significantly lower than that of MRSA patients (38.5%) (P = 0.03). Underlying conditions were similar between BORSA and MRSA patients, but with less dialysis in the former (P = 0.01). Multivariate analysis revealed that septic shock [odds ratio (OR) = 15.95] and bacteraemia originating from lower respiratory tract infection (OR = 5.78) were two independent risk factors for 30-day mortality. The ST45 clone with high-level ciprofloxacin resistance (MICs of 8–128 mg/L) replaced ST59 as the predominant clone since 2012, although no major clustering was detected. In conclusion, the emerging multidrug-resistant ST45 clone has replaced ST59 as the most common among BORSA isolates.

Phenotypic Microarrays Suggest Escherichia coli ST131 Is Not a Metabolically Distinct Lineage of Extra-Intestinal Pathogenic E. coli

Extraintestinal pathogenic E. coli (ExPEC) are the major aetiological agent of urinary tract infections (UTIs) in humans. The emergence of the CTX-M producing clone E. coli ST131 represents a major challenge to public health worldwide. A recent study on the metabolic potential of E. coli isolates demonstrated an association between the E. coli ST131 clone and enhanced utilisation of a panel of metabolic substrates. The studies presented here investigated the metabolic potential of ST131 and other major ExPEC ST isolates using 120 API test reagents and found that ST131 isolates demonstrated a lower metabolic activity for 5 of 120 biochemical tests in comparison to non-ST131 ExPEC isolates. Furthermore, comparative phenotypic microarray analysis showed a lack of specific metabolic profile for ST131 isolates countering the suggestion that these bacteria are metabolically fitter and therefore more successful human pathogens.

Building PulseNet Latin America and Caribbean Salmonella regional database: First conclusions of genetic subtypes of S. Typhi, S. Typhimurium and S. Enteritidis circulating in six countries of the region

PulseNet Latin America and Caribbean Network (PulseNet LA and C) works together with PulseNet International sharing molecular epidemiologic information for the recognition and investigation of foodborne disease outbreaks.The participants of PulseNet LA and C perform standardized pulsed field gel electrophoresis (PFGE) protocols and analysis generating data that is incorporated into Regional Databases. In this study we present the relationship and distribution of genetic subtypes of Salmonella enterica ser. Typhimurium (STM), Salmonella enterica ser. Enteritidis (SE), and Salmonella enterica ser. Typhi (ST) human isolates circulating in six countries of the Region between 2005 and 2009, from the analysis of the Salmonella Database.The 70 ST isolates analyzed were diverse and none of the countries shared the same PFGE profiles with XbaI enzyme. These results show a high genetic diversity among the strains studied and provide background to trace future outbreaks and travel related cases. In the analysis of 550 STM isolates, we found 10 patterns shared at least between two countries, suggesting the need of further studies of attribution to the source of origin. Only one of these PFGE patterns was associated with a known outbreak. Among 225 SE isolates, a predominant subtype was identified, that grouped 83.5% of the isolates and was associated with foodborne outbreaks in five of the six countries; showing the need to use other subtyping techniques for this serovar.The continuous update of PulseNet LA and C Salmonella Regional Database provides an important tool for the laboratory based surveillance of the serovars analyzed, for the prevention and control of foodborne outbreaks, and for the detection of emerging strains in the Region.

Alleviation of the adverse effects of salinity stress in wheat (Triticum aestivum L.) by seed biopriming with salinity tolerant isolates of Trichoderma harzianum

Salt stress is one of the major abiotic stresses limiting crop growth and productivity. This work investigated the potential of five ST isolates of Trichoderma harzianum (Th-13, Th-14, Th-19, Th-33 and Th-50) applied through seed biopriming in reducing the detrimental effects of salinity stress on wheat (Triticum aestivum L.). Growth, physiological and biochemical parameters were studied to characterize salt tolerance. One factor was treatments (T1, T2, T3, T4, T5 and T6) and second factor was four levels of salt stress viz., 0, 2, 4 and 6 dsm−1. In germination test, most of the isolates (Th-14, Th-19 and Th-13) were effective in improving germination percentage and reducing RPG during salinity stress. Seedlings raised from ST Trichoderma isolates had significantly higher root and shoot lengths, CC and MSI than control at all stress levels. The treatments Th-14, Th-19 and Th-13 showed lower accumulation of MDA content whereas proline content and phenolics were higher in treated plants under both non-saline and saline conditions. Highest MDA content was observed in control at salt stress level of 6 dSm−1. It is concluded that seed biopriming with different salinity tolerant isolates of Trichoderma reduced severity of the effects of salinity though the amelioration was better in Th-14 under present experimental material and conditions.

Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples

A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site ( orfX) of the staphylococcal cassette chromosome mec (SCC mec) was evaluated in MRSA SCC mec reference strains ( n = 9 ), MRSA ST strains ( n = 16 ) and clinical isolates of MRSA ( n = 124 ), MSSA ( n = 53 ), methicillin-resistant coagulase-negative staphylococci ( n = 47 ), and methicillin-susceptible, coagulase-negative staphylococci ( n = 32 ). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain–heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.

Prevalence of enterotoxigenic Escherichia coli (ETEC) in hospitalized acute diarrhea patients in Denpasar, Bali, Indonesia

The relationship between enterotoxigenic Escherichia coli (ETEC) and hospitalized patients with acute diarrhea was examined in a study conducted in two hospitals from June 2000 to May 2001 in Denpasar, Bali, Indonesia. A total of 489 hospitalized patients with acute diarrhea were enrolled, and their rectal swabs were screened for enteric bacterial pathogens. Toxins, colonization factor antigens (CFAs), in vitro antimicrobial susceptibility and seasonal distribution patterns associated with ETEC were ascertained. The diagnosis of ETEC infection and CFAs association were performed with GM-1 ELISA and Dot blot immunoassays. Enterotoxigenic Escherichia coli was isolated from the rectal swabs of 14.9% of the patients. The distribution of toxins among the ETEC strains found was ST in 51 (69.9%), while LT and ST/LT were found in 28.8% and 1.3% respectively. The highest isolation rate for ETEC was found among children between the ages of 1 and 15 years. Colonization factor antigens were identified in 28.8% of the ETEC strains. A high prevalence of CFA was found among the rectal swabs of patients with ST isolates. High frequency of resistance to ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, tetracycline and cephalothin was displayed among the ETEC strains. All ETEC strains were susceptible to norfloxacin, ciprofloxacin and nalidixic acid. The results of this study document the prevalence of ETEC in hospitalized patients with acute diarrhea in Denpasar, Bali, Indonesia. Data generated in this study depicts the prevalence of ETEC diarrhea and CFA types among diarrhea patients in the tourist city of Denpasar, Bali, Indonesia.

Beyond serotypes and virulence-associated factors: detection of genetic diversity among O153:H45 CFA/I heat-stable enterotoxigenic Escherichia coli strains.

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like São Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.

The Salmonella ompC gene: Structure and use as a carrier for heterologous sequences

The Salmonella typhi (St) ompC gene codes for a major outer membrane protein (OMP) that is highly expressed in both low and high osmolarity. By hybridization studies with the entire gene or with segments thereof, ompC was found to be highly conserved within 11 different Salmonella serotypes, with the exception of S. arizonae. The study included several St isolates from Mexico and Indonesia. Variation was only detected in two (e and f) of the seven regions previously found to vary between St and E. coli ompC. Chimeric OmpC proteins, carrying a rotavirus VP4 capsid protein epitope, are well recognized by a specific monoclonal antibody (mAb) against this epitope, either in OMP preparations (by enzyme-linked immunosorbent assay; ELISA) or intact cells (by ELISA and immunogold-labelling), indicating that regions c and f are oriented towards the cell surface and are probably exposed. As has been shown before for other regulated OMP, this experimental approach could be useful for the presentation of heterologous epitopes in order to gain knowledge about porin topology, for testing the effect of altered porin surface epitopes on bacterial physiology, or else, in the development of multivalent vaccines.

A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh

We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.