Detection Of Toxin Genes Research Articles

DNA extracted from faeces as a source of information about endemic reindeer from the High Arctic: detection of Shiga toxin genes and the analysis of reindeer male-specific DNA

DNA extracted from faeces may be a valuable source of information about the animal itself, as well as its microflora. An isolated reindeer population from Svalbard (Rangifer tarandus platyrhynchus) was tested for the presence of Shiga toxin encoding genes in the collected faecal samples. Even though the reindeers were not interacting with any other ruminants, which are considered to be a major reservoir of Shiga toxin containing bacteria, the stx1 gene was detected in 9 out of 10 tested samples, and the stx2 subtype c was found in five tested samples. This is an exceptionally high proportion, especially in the case of stx1, when compared to those observed in semi-domesticated or wild cervid populations in less remote locations. Distribution of the investigated genes in a small, local population indicates a different pattern of transmission, which seems to favour bacteria carrying the stx1 genes over those carrying the stx2 genes. The overall high percentage of animals with microbiota containing the stx genes suggests an important role of these genes in such an extreme environment for either hosts or their gut bacteria. Additionally, male-specific DNA found in the faeces was isolated in order to establish a given animal’s sex. PCR based on two pairs of primers, DBY7 and DBY8 gave the expected length of product characteristic for Y chromosome. The results of molecular sexing were consistent with field observations.

Identification of Infantile Diarrhea Caused by Breast Milk-Transmitted Staphylococcus aureus Infection

Staphylococcus aureus is a well-known organism which is responsible for a variety of human infectious diseases including skin infections, pneumonia, bacteremia, and endocarditis. Few of the microorganisms can be transmitted from mother to the newborn or infant by milk breastfeeding. This study aims to identify transmission of S. aureus from healthy, lactating mothers to their infants by breastfeeding. Stool specimens of diarrheal infants and breast milk of their mother (totally three pairs) were collected and six Staphylococcus aureus isolates were cultured positively. Homology and molecular characters of isolated strains were tested using pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing. Furthermore, toxin genes detection was also performed. Each pair of isolates has the same PFGE type and spa type. Four Sequence types (STs) were found among all the isolates; they are ST15, ST188, and ST59, respectively. Among the strains, seb, sec, and tst genes were found, and all were negative for pvl gene. The homology of the S. aureus strains isolated from the infants' stool and the mothers' milk was genetically demonstrated, which indicated that breastfeeding may be important in the transmission of S. aureus infection, and the character of S. aureus needed to be further evaluated.

Detection of Emetic Toxin Genes in Bacillus cereus Isolated from Food and their Production of Cereulide in Liquid Culture

AbstractBacillus cereus is a Gram‐positive that is ubiquitous in foods and the wider environment. The B. cereus emetic toxin, cereulide, synthesized by nonribosomal peptide synthetase (NRPS), can cause either gastrointestinal disorders and/or emetic form of food poisoning. In this study, 108 B. cereus isolates from foods were tested for a correlation between the presence of emetic toxin genes and cereulide production. Based on 16S rRNA sequence analysis, 107 isolates were identical to B. cereus spp. Further experiments showed that fourteen isolates exhibited 1,271, 2,200 and 188‐bp bands, corresponding to amplification of the ces1, ces2 and NRPS gene, respectively. However, only 7 out of 13 strains possessing the NRPS, ces1, and ces2 genes produced cereulide in liquid culture. Strain number 93, isolated from fresh vegetables, produced the highest levels of cereulide (11.17 μg/mL); maximum cereulide levels were observed after 4 days of cultivation at 30C in LB broth. The amount of cereulide produced differed between each isolate.Practical ApplicationsThe incident of Bacillus cereus in food could cause food poisoning. The results of our study on the presence of B. cereus in spoiled foods demonstrated that not all the food poisoning B. cereus spp. have the ability to synthesize the emetic toxin, cereulide. Nonribosomal peptide synthetase and cereulide synthetase genes could be used as biological markers for differentiation of cereulide producing and nonproducing B. cereus. Estimation of cereulide level reveals that the amounts of cereulide produced by some strains were very high and could be a threat to human health if consumed in contaminated foods. This study presents proper methods to select cereulide producing food‐poisoning B. cereus spp. and gives more information on the condition of cereulide production to prevent future cases of emetic food poisoning.

Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients

The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high.

Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays

Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological studies by providing information that could be related to the risk of human infection.

Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification.

Aim:The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens.Materials and Methods:A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene.Results:Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis.Conclusion:Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

The Role of Glutamate Dehydrogenase (GDH) Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China.

The incidence and severity of Clostridium difficile infection (CDI) in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH) test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0%) and specificity (92.8%), compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7%) and positive predictive value (97.2%), but low sensitivity (45.0%) and negative predictive value (88.3%), compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing.

PCR based detection of Alpha toxin gene in Clostridium perfringens strains isolated from diseased broiler chickens

PCR based detection of Alpha toxin gene in Clostridium perfringens strains isolated from diseased broiler chickens

Seasonal Prevalence of Enteropathogenic Vibrio and Their Phages in the Riverine Estuarine Ecosystem of South Bengal.

Diarrheal disease remains an unsolved problem in developing countries. The emergence of new etiological agents (non-cholera vibrios) is a major cause of concern for health planners. We attempted to unveil the seasonal dynamics of entero-pathogenic Vibrios in Gangetic riverine-estuarine ecosystem. 120 surface water samples were collected for a period of one year from 3 sampling sites on the Hooghly river. Five enteropathogenic Vibrio species, V. cholerae (35%), V. parahaemolyticus (22.5%), V. mimicus (19.1%), V. alginolyticus (15.8%) and V. vulnificus (11.6%), were present in the water samples. The vibriophages, V. vulnificus ɸ (17.5%), V. alginolyticus ɸ (17.5%), V. parahaemolyticus ɸ (10%), V. cholerae non-O1/O139 ɸ (26.6%) and V. mimicus ɸ (9.1%), were also detected in these samples. The highest number of Vibrios were noted in the monsoon (20–34°C), and to a lesser extent, in the summer (24–36°C) seasons. Samples positive for phages for any of the identified Vibrio species were mostly devoid of that particular bacterial organism and vice versa. The detection of toxin genes and resistance to β-lactam antibiotics in some environmental enteropathogenic Vibrio species in the aquatic niches is a significant outcome. This finding is instrumental in the south Bengal diarrhoeal incidence.

Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

DNA-Microarray-based Genotyping of Clostridium difficile.

BackgroundClostridium difficile can cause antibiotic-associated diarrhea and a possibility of outbreaks in hospital settings warrants molecular typing. A microarray was designed that included toxin genes (tcdA/B, cdtA/B), genes related to antimicrobial resistance, the slpA gene and additional variable genes.ResultsDNA of six reference strains and 234 clinical isolates from South-Western and Eastern Germany was subjected to linear amplification and labeling with dUTP-linked biotin. Amplicons were hybridized to microarrays providing information on the presence of target genes and on their alleles. Tested isolates were assigned to 37 distinct profiles that clustered mainly according to MLST-defined clades. Three additional profiles were predicted from published genome sequences, although they were not found experimentally.ConclusionsThe microarray based assay allows rapid and high-throughput genotyping of clinical C. difficile isolates including toxin gene detection and strain assignment. Overall hybridization profiles correlated with MLST-derived clades.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0489-2) contains supplementary material, which is available to authorized users.

Detection of cytolethal distending toxin (cdt) Genes in Campylobacter jejuni and Campylobacter coli isolated from the intestinal of commercial broiler chickens, turkey and quail of Iran

Background: Campylobacter jejuni and Campylobacter coli are zoonotic bacteria which are frequently associated with human diarrhea. Sharing of the cytolethal distending toxin (cdt) genes in Campylobacter is common and is considered species specific. OBJECTIVES: In this study we focused on detecting the presence of cdt gene in C. jejuni and C. coli isolated from broilers, turkeys and quails of Iran. METHODS: Cecal samples were randomly collected from 240 broiler chickens, 100 meat type turkeys and 100 quails after slaughtering. We used PCR as a method for detecting cdt genes. RESULTS: In broilers, 93% of 58 C. jejuni positive samples possessed cdt gene and in all cases the three different subunits of cdt genes were present. However, only 56% of 14 C. coli isolates in broilers had contained cdt genes, while one fourth having all three subunits present. In turkeys, around 65% of 34 C. jejuni positive samples had cdt gene present with 38% possessing all three subunits of cdt genes. But all 5 C. coli isolates had all three subunits cdt gene. In quails, 67% of 30 C. jejuni positive samples were identified by cdt gene, 20% of those possessed all three gene subunits. On the other hand, all 28 C. coli isolates of quails had cdt gene present while 36% of those held all three gene subunits. CONCLUSIONS: Our data is indicating the isolation, culture and cdt PCR amplification approaches in this study seemed to be efficient. However, the presence of different variation of Campylobacter cdt gene types in our sample isolates signifies the necessity of further functional gene studies to elucidate which gene type combinations result in encoding effective toxins.

Characterization of Staphylococcus aureus Isolated from Non-Native Patients with Skin and Soft Tissue Infections in Shanghai.

Background Staphylococcus aureus is one predominant cause of skin and soft-tissue infections (SSTIs), but little information exists regarding the characterization of S. aureus from non-native patients with SSTIs in China.MethodsIn this study, we enrolled 52 non-native patients with S. aureus SSTIs, and 65 native control patients with S. aureus SSTIs in Shanghai. 52 and 65 S. aureus isolates were collected from both groups, respectively. S. aureus isolates were characterized by antimicrobial susceptibility testing, toxin gene detection, and molecular typing with sequence type, spa type, agr group and SCCmec type.ResultsMethicillin-resistant S. aureus (MRSA) was detected in 8 non-native patients and 14 native patients with SSTIs. Overall, antimicrobial susceptibilities of S. aureus isolated from non-native patients were found higher than those from native patients. CC59 (ST338 and ST59) was found in a total of 14 isolates (4 from non-native patients; 10 from native patients), 9 of which were carrying lukS/F-PV (3 from non-native patients; 6 from native patients). ST7 was found in 12 isolates and all 12 isolates were found in native patients. The livestock-associated clone ST398 was found in 11 isolates (6 from non-native patients; 5 from native patients), and 5 ST398 lukS/F-PV-positive methicillin-susceptible S. aureus (MSSA) were all discovered among non-native patients. The molecular epidemiology of S. aureus isolated from non-native patients was quite different from those from native patients. lukS/F-PV was more frequent in isolates originating from non-native patients with SSTIs compared to native patients (31 vs. 7, P <0.0001).ConclusionsCC59 was the most common clonal complex among patients with SSTIs in Shanghai. The other most common sequence types were ST7 and Livestock ST398. The molecular epidemiology of S. aureus isolated from non-native patients was quite different from those from native patients. S. aureus isolated from non-native patients was more likely to carry lukS/F-PV.

Detection of cytolethal distending toxin (cdt) genes of Campylobacter Jejuni and Coli in fecal samples of pet birds in Iran

BACKGROUND: Campylobacteriosis is one of the most commonbacterial intestinal disorders of humans in many countries.A wide range of domestic and wild bird species have been identifiedas natural reservoirs. OBJECTIVES: The aim of this studywas to determine the Campylobacter spp from pet birds andscreen the determined Campylobacters for presence of virulencecytolethal distending toxin (cdt) genes. METHODS: A totalof 660 fecal samples from 32 different species of pet birds weretaken and examined for detection of Campylobacter spp. andwere investigated for presence of cdt genes. All the sampleswere collected from clinically healthy birds that were kept incage, zoological parks, and/ or in zoo of Tehran, the capital ofIran. RESULTS: In total, 20 thermophilic Campylobacter weredetected from 8 different avian species. From 20 confirmedCampylobacter spp., 16 samples (80%) were C. jejuni positiveand 4 (20%) were C. coli in species-specific PCR test. Furthermore,out of 20 detected Campylobacter, 13 (65%) harboredthe various subunits of cdtA, cdtB and cdtC genes, and 7 (35%)were negative for all tested cdt genes. CONCLUSIONS: Our findingsindicate that the carriage rate of Campylobacter in differentspecies of cage and/or in zoo birds is high and confirm thatcdt genes may frequently be present in campylobacter spp.

Phenotypic and phylogenetic analysis of Vibrio parahaemolyticus isolates recovered from diarrhea cases in Guangdong Province, China

Vibrio parahaemolyticus has emerged as a common foodborne pathogen of global concern. In this study, 108 V. parahaemolyticus isolates that recovered from diarrhea cases (n=96) and seafood products (n=12) in Guangdong Province from 2007 to 2011 were characterized by serotyping, tdh and trh toxin gene detection and multilocus sequence typing (MLST). The dominant serotypes from the cases were O3:K6, O4:K8 and O1: KUT (untyped). However, most isolates recovered from seafood products belonged to other serotypes. None of the isolates carried the trh gene, while the major isolates from the cases were tdh positive. MLST analysis revealed 31 sequence types (STs); 17 STs were unique in this study. eBURST analysis revealed four clonal complexes (CC), The majority of the isolates (n=58, all from cases and tdh+) were grouped into the CC3, which included O3:K6, O4:K68 and O1:KUT isolates. The CC3 was the most prevalent clonal complex, and all of the CC3 isolates were recovered from clinical cases of geographically diverse origin. As to the CC345, which was completely constituted by O4:K8, was another important clonal complex affecting Guangdong Province. Ongoing surveillance of V. parahaemolyticus in diarrhea patients and seafood products remains a public health priority for Guangdong Province, China.

Detection of toxin genes and RAPD analysis of bacillus cereus isolates from different soil types

The aim of this study was to detect genes for enterotoxins (hbla, entFM and bceT) and for emetic toxin (cer), to determine antibiotic resistance, and to estimate intraspecies diversity in B. cereus isolates by RAPD analysis. B. cereus was identified in 12 out of 117 indigenous Bacillus spp. using the classical microbiological methods and PCR. All isolates were resistant to penicillin and ampicillin, two to tetracyclin and four to trimethoprim-sulphamethoxazole. Also, all isolates produced inducible penicillinases and ?-lactamase. Toxin genes were detected with PCR. EntFM and cer genes were present in all isolates, hbla in all, but two, and bceT in none. RAPD analysis was performed with four different primers, two of them designed for this study. The intraspecies diversity revealed 10 different patterns at the 90% similarity level. Two separate clusters were formed regardless of a soil type or utilization. The detection of genes encoding toxins in all B. cereus isolates indicated these bacteria as potentially pathogenic and seriously for human health. Regardless of a soil type or utilization, the RAPD analysis showed high intraspecies heterogeneity in B. cereus isolates. To the best of our knowledge, this is the first study to analyse the presence of entero- and emetic toxin genes and genetic heterogeneity in B. cereus isolates from different soil types and different soil utilization in Serbia.

A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli

Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30–80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the “Top-6” serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic genotypes as demonstrated in this paper is rapid, easily performed, informative and cost-effective, and thus has a potential to be deployed in the food industry for the routine screening of potentially pathogenic STEC isolates.

Characteristics of qacA/B-positive Staphylococcus aureus isolated from patients and a hospital environment in China.

This study was designed to demonstrate the characteristics of qacA/B-positive Staphylococcus aureus in China. One hundred and forty-five MRSA and 178 MSSA from clinical specimens from seven hospitals in different regions of China, 70 MRSA from superficial sites of patients and 106 MRSA from environmental samples from an ICU were collected and screened for the presence of the qacA/B gene. The qacA/B-positive isolates and 72 randomly selected qacA/B-negative control isolates were further characterized by MLST, spa typing and detection of toxin genes, as well as antimicrobial and chlorhexidine susceptibility. SCCmec typing was conducted for MRSA. PFGE was conducted for qacA/B-positive isolates. Twenty-five (7.8%) of the 321 MRSA isolates harboured qacA/B, including 11 isolates from clinical specimens (7.6%), 12 isolates from patients' superficial sites (17.1%) and 2 isolates from an ICU environment (1.9%). Ten and five qacA/B-positive MRSA were identified as ST239-t030-MRSA-III and ST239-t037-MRSA-III, respectively. Six PFGE clusters and five singletons were identified among the 25 qacA/B-positive MRSA. Only one (0.6%) of the 178 MSSA isolates harboured qacA/B. qacA/B carriage in MRSA was statistically associated with spa-t037 and the presence of mupA. Compared with qacA/B-negative MRSA, the qacA/B-positive MRSA exhibited a lower susceptibility to chlorhexidine and higher resistance rates to clindamycin and trimethoprim/sulfamethoxazole. Carriage of qacA/B, although it had a low prevalence, might be the main reason for declining susceptibility to chlorhexidine in MRSA from Chinese patients and is probably associated with spa-t037 and the presence of the mupA gene.

Preponderance of toxigenic Escherichia coli in stool pathogens correlates with toxin detection in accessible drinking-water sources.

Since early detection of pathogens and their virulence factors contribute to intervention and control strategies, we assessed the enteropathogens in diarrhoea disease and investigated the link between toxigenic strains of Escherichia coli from stool and drinking-water sources; and determined the expression of toxin genes by antibiotic-resistant E. coli in Lagos, Nigeria. This was compared with isolates from diarrhoeal stool and water from Wisconsin, USA. The new Luminex xTAG GPP (Gastroplex) technique and conventional real-time PCR were used to profile enteric pathogens and E. coli toxin gene isolates, respectively. Results showed the pathogen profile of stool and indicated a relationship between E. coli toxin genes in water and stool from Lagos which was absent in Wisconsin isolates. The Gastroplex technique was efficient for multiple enteric pathogens and toxin gene detection. The co-existence of antibiotic resistance with enteroinvasive E. coli toxin genes suggests an additional prognostic burden on patients.

English

The formation of biofilms on pieces of equipment and dental chair unit waterlines has attracted more interest and concern. This study aimed to assess the reliability of two phenotypic methods used to detect the presence of biofilms and the icaADBC operon, and identify various toxin genes by multiplex polymerase chain reaction (PCR) in clinical staphylococcal isolates. A total of thirty-one (31) staphylococcal strains were isolated from waterline tubings in the dental unit. Two phenotypic methods were used for the detection of biofilm production. PCR detected the presence of the ica operon, and a multiplex PCR assay for the detection of staphylococcal toxin genes was done. Biofilm production was detected in twenty-six (83.87%) isolates by tissue culture plate method (TCP). The CRA method indicated that twenty (64.51%) of these strains could produce slime. By using the ica operon test, the genotypic ability to form biofilm in was identified in fourteen (45.16%) strains. Five (16.12%) strains of Staphylococcus warneri harbored one or more toxin genes detected by PCR multiplex. The presence of ica operon gene does not always have relationship with in vitro biofilm formation and genes encoding the staphylococcal toxin. Among the strains studied, few of them had the ica operon, toxin gene, and phenotypic ability to form a biofilm at the same time. Key words: Dental unit waterlines, staphylococci, biofilm, Ica genes, toxin genes.