Toxin Gene Profiles Research Articles

Activity of spore-crystal mixtures of new Bacillus thuringiensis strains against Dendrolimus pini (Lepidoptera: Lasiocampidae) and Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract We estimated the usefulness of spore-crystals preparations of the two B. thuringiensis isolates, MPU B9 and MPU B54, for reducing the number of pests. The potential insecticidal toxicities of B. thuringiensis isolates were assessed by the analysis of the genes coding for crystalline proteins. The activities of spore-crystals preparations were determined against Dendrolimus pini L. (Lepidoptera: Lasiocampidae) and compared with the toxicity of spores and crystals of B. thuringiensis subsp. kurstaki HD-1 from commercial biopesticide Foray. Although the analysis of crystalline toxin gene profiles indicated potentially higher activities of MPU B9 and MPU B54 crystals against the pests than that of HD-1, the toxicities of isolate and HD-1 preparations against D. pini caterpillars were similar. The LC50 amounted to 3.42×104 spores and crystals for HD-1, 3.36×104 for MPU B9 and 3.5×104 for MPU B54. Additionally, the toxicity of the MPU B54 preparation was evaluated against Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae). The LC50 was 4.5×105 spores and crystals of MPU B54, and 2.69×106 spores and crystals of HD-1. The LC50 of the MPU B54 preparation against S. exigua was approximately six-fold higher than that of HD-1. However, due to the very wide fiducidal limits for LC50 values, which for both preparations overlap to a large extent, the toxicity of the preparations should be considered the same. The varied profiles of crystalline toxin genes and important toxicity of spore-crystal mixtures of isolates against S. exigua and D. pini indicate the effectiveness of the mixtures against pests and make the strains an alternative for HD-1 for reducing the number of insects.

Molecular Investigation of Methicillin-Resistant Staphylococcus aureus Strains Recovered from the Intensive Care Unit (ICU) Based on Toxin, Adhesion Genes and agr Locus Type Analysis

Background: Staphylococcus aureus, as one of the most common causes of nosocomial infections, has widely spread to all parts of the world and is becoming a serious concern in public health. Objectives: The present study aimed at evaluating the prevalence of adhesion and toxin gene profiles and their distribution among different agr types. Methods: The current cross-sectional study was performed in Tehran, Iran, by analyzing 125 methicillin resistant Staphylococcus aureus (MRSA) strains isolated from hospitalized patients at the ICUs from March, 2016 to January, 2017. In vitro antibiotic susceptibility testing of isolates was assessed using the Kirby-Bauer disk diffusion method. The MRSA strains were genetically typed by agr typing and virulence and adhesion genes profile by conventional PCR. Results: Antibiotic susceptibility testing showed that inducible macrolide-lincosamide-streptogramin B, constitutive macrolide-lincosamide-streptogramin B, and high-level mupirocin resistance phenotypes had a frequency of 18 (14.4%), 50 (56%), and 10 (31.3%), respectively. The predominant resistance profile among MDR-MRSA isolates included resistance profile to seven antibiotics (32%). A total of ten virulence genotypes were observed, from which genotype spa, clfA, clfB, fnbB, fnbA, ebp, and can / tst (36%, 45/125) comprised the majority followed by spa, clfA, clfB, and fnbB (24%, 30/125). Type I was the most prevalent agr type (52%), followed by type III (34.4%), type II (9.6%), I 5(5.3%), and IV (4%). All isolates carrying PVL-encoding genes and HLMUPR-MRSA strains corresponded exclusively to agr type I. Conclusions: The current data demonstrated that virulence gene profiles among different agr types of MRSA isolates were divers. The present study suggests that molecular characterization of MRSA strains should periodically be studied.

Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus from Retail Raw Chicken Meat

The aim of this study was to investigate the toxin gene profile and antimicrobial resistance of Staphylococcus aureus isolates from raw chicken in the People's Republic of China. In total, 289 S. aureus isolates were characterized by antimicrobial susceptibility testing, and genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin, and toxic shock syndrome toxin were revealed by PCR. Overall, 46.0% of the isolates were positive for one or more toxin genes. A high proportion of toxin genes were pvl (26.6%), followed by sej (12.5%), sea (9.0%), seh (8.3%), seb (6.9%), sec (6.9%), sed (4.8%), sei (3.1%), and see (2.4%). None of the isolates harbored seg, tsst-1, or exfoliative toxin genes. In total, 29 toxin gene profiles were obtained, and pvl (10.7%) was the most frequent genotype, followed by sea (5.9%), seb (4.8%), and sej (4.2%). Furthermore, 99.7% of the strains were resistant to at least one of the tested antimicrobial agents, and 87.2% of them displayed multidrug resistance. Resistance was most frequently observed to trimethoprim-sulfamethoxazole and erythromycin (86.2% for each), followed by tetracycline (69.9%), amoxicillin-clavulanic acid (45.0%), and ampicillin (42.6%). None of the strains were resistant to vancomycin. This study indicates that S. aureus isolates from raw chicken harbored multiple toxin genes and exhibited multiple antimicrobial resistance, which represents a potential health hazard for consumers.

Molecular Epidemiology and Virulence Features of Staphylococcus aureus Bloodstream Isolates in a Regional Burn Center in China, 2012-2016.

Staphylococcus aureus is known to be a predominant pathogen causing bloodstream infection (BSI) from burn units. Our study aimed to perform the clinical epidemiological analysis and virulence features of S. aureus strains isolated from the burn patients with BSI from a burn center in southeastern China during 2012-2016. A collection of 112 S. aureus isolates causing BSI from burn center of a tertiary care hospital in China was carried out. Antimicrobial susceptibility testing was conducted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Toxin gene profiles, multilocus sequence typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) locus typing, staphylococcal cassette chromosome mec (SCCmec) typing, and dendrographic analysis were used to characterize and analyze these isolates. Of 112 S. aureus isolates, 52 (46.4%) were methicillin-resistant S. aureus (MRSA) and 60 (53.6%) were methicillin-susceptible S. aureus (MSSA). ST239-SCCmec III-t030-agr I was the major prevalent clone (26 from MRSA and 6 from MSSA), which was followed by ST239-SCCmec III-t037-agr I (12, 10.0%) and ST5-SCCmec II-t002-agr I (11, 9.2%). The genotyping results showed high genetic diversity in molecular characterization and toxin gene profiles of the strains. Carriage of tsst-1 was mainly associated with ST239-SCCmec III-t030-agr I and ST30-SCCmec IV-t062-agr III, whereas lukS/F-PV was distributed in different clones. In conclusion, ST239-SCCmec III-t030-agr I is the commonest clone causing BSI among burn patients in eastern regions of China. In contrast to MRSA, polyclonality was statistically significantly higher among MSSA isolated from burn patients with BSI.

Genetic diversity and toxin gene distribution among serovars of Actinobacillus pleuropneumoniae from Australian pigs.

To explore the diversity among isolates of the Actinobacillus pleuropneumoniae serovars most common in Australia (serovars 1, 5, 7 and 15) and to examine the Apx toxin profiles in selected representative isolates. A total of 250 isolates selected from different farms were examined for their genotypic profiles and a subset of 122 isolates for their toxin profiles. The isolates of serovars 1, 5, 7 and 15 selected for this study came from different farms and different Australian states and were submitted for serotyping to the reference laboratory. The overall diversity of the strains was explored with the enterobacterial repetitive intergenic consensus (ERIC) PCR and the presence of the toxin genes was investigated with a toxin PCR assay. Some degree of variation was observed in the ERIC-PCR pattern within all four serovars, ranging from 38% to 61% genetic diversity. When looking at the toxin gene profile and, therefore, the predicted ability to produce the expected toxin pattern, one isolate each of serovars 1 (n = 20) and 7 (n = 47) and 17 isolates of serovar 15 (n = 40) showed variation to the expected gene profile. The variations in toxin gene patterns, as detected by PCR, found in this study could be related to significant changes in the gene sequence or total absence of the gene. Variation in toxin gene sequences has been observed in other countries. This variation in the toxin profile could also explain possible variation in pathogenicity observed in the field.

Prevalence, toxin gene profile, antibiotic resistance, and molecular characterization of Clostridium perfringens from diarrheic and non-diarrheic dogs in Korea.

Clostridium perfringens causes diarrhea and other diseases in animals and humans. We investigated the prevalence, toxin gene profiles, and antibiotic resistance of C. perfringens isolated from diarrheic dogs (DD) and non-diarrheic dogs (ND) in two animal hospitals in Seoul, Korea. Fecal samples were collected from clinically DD (n = 49) and ND (n = 34). C. perfringens was isolated from 31 of 49 DD (63.3%) and 21 of 34 ND dogs (61.8%). All C. perfringens strains were positive for the α toxin gene, but not for the β, ε, or ι toxin genes; therefore, all strains were identified as type A C. perfringens. All isolates were cpe-negative, whereas the β2 toxin gene was identified in 83.9% and 61.9% of isolates from DD and ND, respectively. Most isolates were susceptible to ampicillin (94%), chloramphenicol (92%), metronidazole (100%), moxifloxacin (96%), and imipenem (100%). However, 25.0% and 21.2% of isolates were resistant to tetracycline and clindamycin, respectively. Molecular subtyping of the isolated strains was performed by using pulsed-field gel electrophoresis. Fifty-two isolates were classified into 48 pulsotypes based on more than 90% similarity of banding patterns. No notable differences were observed among the isolates from DD and ND.

Development of Multiplex PCR for Staphylococcal Enterotoxin Gene typing and Detection of Enterotoxigenic Staphylococcus aureus

Staphylococcus enterotoxins (SEs) are the main etiological agents of staphylococcal food poisoning. They were classified by serological criteria into 5 major groups and recently many more groups were identified. Staphylococcus enterotoxins (SEs) are pyrogenic toxins of the super-antigen family. Therefore to ensure food safety, it is important to detect and quantify the toxin genes to confirm the presence of enterotoxigenic S. aureus in any food samples. In this study, we have developed a multiplex PCR method to profile major toxin genes such as seB, seD, seE, seI and seM of Staphylococcus aureus using reference strains as well as some food isolates. The sensitivity of the single target and mixture of toxin gene targets were tested from five-fold serially diluted respective genomic DNA samples. The single PCR and multiplex PCR assays can successfully amplify the genomic DNA when used template as low as 4.5 pg/µl and 900 pg/µl respectively. Further these primers were validated on other species including target-negative reference strains as well as other gram positive/ negative reference strains. Among tested food isolates, 66% of them found to be positive for seB and 53% of the food isolates found to have seB and seE genes together. This method is highly suitable for convenient toxin gene profiling and to detect the presence of enterotoxigenic S. aureus.

Distribution of Genes Encoding Toxin, Adhesion, and Antibacterial Resistance Among Various SCCmec Types of Methicillin-Resistant Staphylococcus aureus Isolated From Intensive Care Unit, Tehran, Iran

Background: Most methicillin-resistant Staphylococcus aureus (MRSA) infections occur in health care settings; therefore, it is important to know about antimicrobial susceptibility, and carriage of virulence genes in S. aureus isolates. Objectives: The current study aimed at determining the prevalence of genes coding antimicrobial resistance, toxins, and adhesion factors among various SCCmec types of MRSA isolated from intensive care units (ICUs). Methods: From April 2016 to March 2017, a total of 200 MRSA species were isolated from various clinical samples of patients hospitalized in ICUs. The Kirby-Bauer disk diffusion method was employed to determine resistance pattern. Conventional polymerase chain reaction (PCR) assay was utilized to demonstrate antimicrobial resistance, toxins, and adhesion genes. Different SCCmec types of MRSA strains were determine by the multiplex PCR assay. Results: Antibiotic susceptibility testing revealed that all the isolates were sensitive to linezolid, teicoplanin, and vancomycin. The frequency of high-level mupirocin-resistant MRSA was 5.5%. The presence of resistance genes ant(4’)-Ia, aac(6’)-Ie/aph(2’’), tetM, msrA, ph(3’)-IIIa, ermA, msrB, ermB, ermC, and mupA were detected 73.5%, 60.5%, 57.5%, 37%, 36.5%, 34.5%, 24%, 17%, 15% and 5.5%, respectively. The most prevalent adhesion genes were clfA (93.5%) followed by clfB (90%), fnbA (81.5%), fnbB (77%), can (51%), ebp (46.5%), and bbp (2.5%). The frequency of etb, eta, pvl, and tst genes were 4.5%, 9.5%, 21.5%, and 61.5%, respectively. Inducible macrolide-lincosamide-streptogramin B (inducible MLSB resistance; iMLSB), and constitutive MLSB (cMLSB) phenotypes were observed in 88 (44%) and 26 (13%) isolates. Different SCCmec types comprising type III (56.5%), type IV (25%), type II (11%) and type I (7.5%) were identified among the MRSA strains. Conclusions: Results of the current study showed a high prevalence of resistance to commonly used antibiotics that can be a serious threat to patients hospitalized in ICUs. Also, the current study findings showed that different SCCmec types causing nosocomial infections were associated with specific antimicrobial resistance, adhesion, and toxin gene profiles.

Prevalence of binary toxin positive Clostridium difficile in diarrhoeal humans in the absence of epidemic ribotype 027.

Virulence of Clostridium difficile is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of C. difficile possessing only CDT genes (A−B−CDT+) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of C. difficile has been suggested previously. Most human diagnostic tests will not detect A−B−CDT+ strains of C. difficile because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A−B−CDT+ C. difficile in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (n = 43) were cultured by enrichment, and C. difficile PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 C. difficile isolates were recovered, including two A−B−CDT+ isolates. This corresponded to an A−B−CDT+ prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A−B− isolates, 2/3 CDT+ isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated C. difficile was found. Neither A−B−CDT+ isolate was the predominant A−B−CDT+ strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A−B−CDT+ C. difficile in patients and strain collections but the prevalence of human A−B−CDT+ C. difficile is rarely investigated. This study highlights the occurrence of A−B−CDT+ strains of C. difficile in symptomatic patients, warranting further investigations of its role in human infection.

Clostridium difficile Molecular Epidemiology in a Prospective Cohort of Canadian Children Compared with Cases of C. difficile Infection

Background Clostridium difficile is a notorious nosocomial pathogen, but little is known regarding the colonization commonly observed in children. It is suspected that C. difficile carriage in infants is a reservoir for toxigenic strains. To test this hypothesis, we sought to determine the genetic relatedness between a prospective cohort of C. difficile toxin gene positive healthy children and those with acute gastroenteritis (AGE) and strains identified in adult and pediatric C. difficile infection (CDI) cases from Alberta, Canada. Additionally, we compared C. difficile toxin production in healthy and AGE children.Methods C. difficile was cultured from 97 hospitalized CDI cases (n = 79 adult; n = 18 pediatric) from stool samples tested positive for toxigenic C. difficile by C.DIFF QUIK CHEK COMPLETE® enzyme immunoassay (EIA) in 2015 and samples tested positive for toxin genes by the Luminex xTAG® Gastrointestinal Pathogen Panel from a prospective cohort of 59 children with AGE seeking care at the emergency department and 17 healthy children attending public health clinics. Isolates were then characterized by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), PCR of the tcdA, tcdB, tcdC, and cdtB genes and C. difficile toxigenicity by EIA for a subset of 14 healthy and 45 AGE children.ResultsRibotype 106 was predominant among all pediatric isolates (n = 21, 27.6% AGE and healthy children; n = 5, 27.8% pediatric CDI) and ribotype 027 in adult CDIs (n = 35, 44.3%). Eighteen ribotypes were shared between children and CDI cases (n = 134, 77.5%). Sixteen unique ribotype and PFGE patterns (n = 84, 48.6%) were identified in two or more cohorts. Similar toxin gene profiles were observed across the three cohorts, but adult CDI isolates had a higher proportion of binary toxin positive isolates (n = 42, 53.2%) compared with children (n = 3, 3.95%) and pediatric CDI (n = 0). C. difficile toxigenicity was similar (P = 0.23) amongst the subset of healthy (n = 6, 42.9%) and AGE (n = 28, 62.2%) children.ConclusionProduction of C. difficile toxins in children was not significantly associated with symptoms of AGE. C. difficile strains found in children were similar to those from CDI cases; especially pediatric cases. This suggests that strains might be shared, but the development of CDI may be related to factors other than C. difficile strain type.Disclosures All authors: No reported disclosures.

Molecular detection of enterotoxigenic Staphylococcus aureus isolated from mutton marketed in retail outlets of Chennai, India

The present study was aimed at detection of enterotoxigenic S. aureus in mutton marketed in retail outlets of Chennai. A total of 120 meat samples were collected from across Chennai for isolation of S. aureus and it was observed that 66.28 per cent of the samples were contaminated with S. aureus. The S. aureus count in mutton samples ranged from 1.8 x 102 to 4.9 x 104 CFU/ g with an overall average of 1.30 x 104 CFU/ g. All the isolates presumptively identified as S. aureus biochemically, amplified 181 bp product specific for nuc gene by PCR, which is species specific marker for S. aureus. Enterotoxin gene profiles (multiplex PCR) results revealed that 70.17 percent of the isolates were enterotoxigenic carrying only six genes (seb, sed, seg, seh, sei and sej) either alone or in combination, whereas none of these isolates harbored sea, sec and see. It was clear that seb (72.5 %) was the predominant enterotoxin gene followed by seg and sei, seh, sej and sed. Six different toxin gene profiles were exhibited by different isolates and majority of the isolates (55 %) carried two or more genes as compared to only one toxin gene.

Genotypes and the persistence survival phenotypes of Bacillus cereus isolated from UHT milk processing lines

Abstract: Bacillus cereus is an important foodborne pathogen that can cause emesis and diarrhea. The aim of this study was to find the phenotypic properties of Bacillus cereus enabling its persistence in dairy plants, and identify the corresponding genotypic features. The toxin gene profile, multilocus sequence typing (MLST), biofilm-forming capability and the alkali and acid tolerance of the biofilms of the strains were analyzed. All strains were positive for one or more toxin genes tested, except for A20. The three main toxin genes were nheABC (74.07%), bceT (73.37%), and ces (48.15%), followed by entFM (40.74%) and cytK (33.33%), but no strain harbored hblABD . A total of 17 ST-types were generated among 27 isolates by MLST, and clustered into four groups. Although no significant difference was found in the tolerance of biofilm to acid and alkali among four MLST groups, the strains of MLST groupⅠhad stronger biofilm formation capability. The biofilm was formed on stainless steel coupons, of which the OD values of A1, B16 and B18 were more than 1.0, and close to each other in the phylogenetic tree. Persistence survival strategies analysis showed that strains can be divided into three groups: 15 strains without persistence strategies; two strains were capable of forming biofilm may be effectively inactivated by hot-acid or hot-alkali; ten isolates were capable of forming biofilm and possessing the tolerance of biofilm to acid and alkali, which should be under strict supervision and control in the future.

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cry1, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027.

To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.

Genetic diversity, antimicrobial resistance, toxin gene profiles, and toxin production ability of Bacillus cereus isolates from doenjang, a Korean fermented soybean paste

AbstractThe objectives of this study were to determine the toxin gene profile, toxin production, and antibiotic resistance of Bacillus cereus isolates from doenjang, a Korean fermented soybean paste. Repetitive element sequence polymorphism‐PCR (rep‐PCR) was also used to assess the genetic diversity between these isolates. Thirty‐five B. cereus strains were isolated from 67 commercial doenjang samples, and all 35 isolates carried the nheABC and entFM genes and produced NHE enterotoxin. Of them, 12 strains harbored the emetic toxin gene (ces) and tested positive for the HEp‐2 cell emetic toxin assay. The B. cereus isolates were highly resistant to ß‐lactam antibiotics including ampicillin, cefepime, penicillin, and oxacillin. The rep‐PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes or antibiotic resistance profiles. However, rep‐PCR assay can be used to discriminate emetic B. cereus isolates from nonemetic isolates.Practical applicationsDoenjang has been the focus of recent concern in Korea because of its potential to cause foodborne illness via Bacillus cereus. The results of the present study suggest that B. cereus isolates from doenjang could cause either diarrheal or emetic types of foodborne illness because all isolates contained at least one toxin and its gene, although B. cereus levels in doenjang meet the Korean Food and Drug Administration guidelines (&lt;104 cfu/g). Thus, it is necessary to constantly monitor the levels of B. cereus contamination and its toxins in doenjang.

Analysis of toxin and multilocus sequence typing of Clostridium difficile strains isolated from China-Japan Friendship Hospital

Objective To analyze the characteristics of toxin, the PCR-ribotyping(RT) and the multilocus sequence typing(MLST) of Clostridium difficile strains isolated from China-Japan Friendship Hospital in order to provide a basis for monitoring the outbreak of nosocomial Clostridium difficile infection. Methods A total of 321 samples were collected from the patients with suspected Clostridium difficile infection(CDI) in China-Japan Friendship Hospital(CJFH) during 2012 to 2013. All Clostridium difficile strains were isolated and identified by the standard phenotypic culture method. Cytotoxicity test was performed to detect toxin B. Toxin genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) harbored by those strains were analyzed. RT and MLST were used for homologous analysis. Clinical data of the patients were collected to analyze the isolation rate of Clostridium difficile in different populations. Results Forty-eight strains of Clostridium difficile were isolated from 46 patients with diarrhea and three of them were isolated from the same patient. The incidence of CDI among all patients, outpatients and inpatients were 14.3%(46/321), 12.8%(5/39) and 14.5%(41/282), respectively. Toxin B was detected in all of the strains as indicated by the cytotoxicity test. Strains of sequence type 1(ST1) showed the strongest cytotoxicity of all the isolated Clostridium difficile strains. Ten out of the 48 strains (20.8%) were tcdA(-)/tcdB(+ ) strains, which belonged to either ST37 or ST81. The results of RT and MLST were consistent in assigning the strains into nine types, in which the predominant type was ST1/RT027 accounting for 27.1% (13/48). All of the ST1/RT027 strains presented a toxin gene profile of tcdA(+ )/tcdB(+ ) and cdtA(+ )/cdtB(+ ). Most of the ST1/RT027 strains were isolated from the Traditional Chinese Medicine Department of Respiratory, where smallnosocomial outbreaks of ST1/RT027 strain infection might happen. Conclusion CDI diagnosed in CJFH mainly belongs to nosocomial infection. Most of the isolated strains harbor tcdA(+ )/tcdB(+ ) genes. Surveillance for the outbreaks of CDI caused by ST1/RT027 strains over producing toxins A and B should be strengthened in hospitals. Key words: Clostridium difficile; Standard phenotypic culture method; Cytotoxicity test; Ribotyping; Multilocus sequence typing

Relationships among superantigen toxin gene profiles, genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis.

The prevalence, genetic diversity and antibiotic resistance of Staphylococcus aureus in milk, whey, and cheese from artisan farm dairies

In this study, coagulase positive staphylococci were detected in 45% of the 69 bovine milk, whey and cheese samples taken from five farm dairies, and all raw milk samples were contaminated. Genetic diversity, staphylococcal enterotoxin genes and antimicrobial susceptibility in putative Staphylococcus aureus isolates were investigated. Sixty-one percent of the 72 isolates analysed belonged to the same pulsed field gel electrophoresis group. The spa-typing revealed seven different spa types, t2678 being the most prevalent, but t127 and t197 were also detected. Sixteen different toxin gene profiles were identified in 87.5% of the isolates with sec and tst being the most frequent (52.5%), followed by seg and seh. All isolates were methicillin-sensitive S. aureus, and sensitive to the 12 antibiotics tested. The prevalence of S. aureus and the high diversity of isolates carrying enterotoxin genes constitute grounds for food safety concern in artisanal cheese making, whether pasteurised or not.

Staphylococci isolated from ready-to-eat meat – Identification, antibiotic resistance and toxin gene profile

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance.

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.