Enterobacterial Repetitive Intergenic Consensus Sequence Research Articles

Serotype Distribution, Antimicrobial Resistance, Virulence Genes, and Genetic Diversity of Salmonella spp. Isolated from small-scale Leafy Green Vegetable Supply Chains in South Africa

Salmonella have been implicated in foodborne disease outbreaks globally and is a pressing concern in the South African small-scale sector due to inadequate hygiene standards and limited regulatory oversight, leading to a higher risk of foodborne diseases. By investigating irrigation water and leafy green vegetables produced by small-scale growers and sold through unregulated supply chains, this study was able to determine the presence, serotype distribution, virulence gene profiles, antibiotic resistance, and genetic diversity of Salmonella isolated from these sources. From 426 samples, 21 Salmonella-positive samples were identified, providing 53 Salmonella isolates. Of these, six different Salmonella serotypes and sequence types (STs) were identified, including Salmonella II 42:r: ST1208 (33.96%; n = 18), Salmonella Enteritidis: ST11 (22.64%; n = 12), Salmonella II 42:z29: ST4395 (16.98%; n = 9), Salmonella Havana: ST1524 (15.09%; n = 8), Salmonella Typhimurium: ST19 (9.43%; n = 5), and Salmonella IIIb 47:i:z: ST7890 (1.89%; n = 1). A total of 92.45% of the isolates were found to be multidrug-resistant, showing high rates of resistance to aztreonam (88.68%; n = 47), ceftazidime (86.79%; n = 46), nalidixic acid (77.36%; n = 41), cefotaxime (75.47%; n = 40), cefepime (71.70%; n = 38), and streptomycin (69.81%; n = 37). All isolates possessed the aac(6')-Iaa antimicrobial resistance gene, with a range of between 9 and 256 virulence genes. Eleven cluster patterns were observed from Enterobacterial Repetitive Intergenic Consensus sequence analyses, demonstrating high diversity among the Salmonella spp., with water and fresh produce isolates clustering, suggesting water as a potential contamination source. Plasmid replicon types were identified in 41.51% (n = 22) of the isolates, including Col(pHAD28) in Salmonella Havana (5.66%; n = 3), Col156 in Salmonella II 42:z29:- (1.89%; n = 1) and both IncFIB(S) and IncFII(S) in Salmonella Enteritidis (22.64; n = 12), Salmonella Typhimurium (9.43%; n = 5), and Salmonella Havana (1.89%; n = 1). This study highlights the presence of multidrug-resistant and multivirulent Salmonella spp. in the small-scale leafy green vegetable supply chains, underscoring the need for the development of a “fit-for-purpose” food safety management system within this system.

Occurrence and molecular characterization of Salmonella Typhimurium and Salmonella Enteritidis isolates from contaminated food samples from Palestine

Salmonella is one of the most frequently isolated foodborne pathogens. It is of major public health concern worldwide. Poultry meat and eggs represent an important source of Salmonellae organism for consumer health. This study aimed to evaluate the occurrence of Salmonella enterica serotype Typhimurium and Enteritidis using multiplex PCR (mPCR) among isolates collected from the local market and to assess genetic relationships between isolates of S. Typhimurium, which was the only serotype isolated from the tested food samples. This was done using virulence factors profiling and fingerprint profiling by random amplified polymorphic DNA (RAPD-PCR) and repetitive sequence PCR (REP-PCR) using enterobacterial repetitive intergenic consensus (ERIC-PCR) and interspersed repetitive DNA sequence BOXAIR-PCR.
 The overall occurrence percentage of S. Typhimurium and S. Enteritidis out of 51 isolates was 54.9% and 0.0%, respectively. Only 13 out of 17 virulence genes were detected in these isolates. The occurrence of the detected virulence genes among these isolates was 100%, 50.0%,46.4%, 39.3%, 35.7%, 35.7%, 32.1%, 25.0%, 25.0%, 17.6%, 14.3%, 14.3%, 3.6% for invA, sopB, prgH, sitC, pefA, tolC, cdtB, msgA, sifA, iroN, spiA, ipfC and pagC, respectively. The remaining virulence genes were absent in all of the isolates. Based on the combination of the presence and absence of virulence genes, eight profiles were detected among these isolates, the most common genetic profile was V5 (each 32.1%). Based on this genetic profile at cut-off point 96.0%, both ERIC and BOX primers allowed for discrimination into 4 and 6 clusters or clones of 16 S. Typhimurium isolates, respectively. Results of PCR typing methods showed that, three strains clustered together using both ERIC-PCR and BOX-PCR typing methods and they had the same virulotype (V1), while other four strains also clustered together by both typing methods and had the same virulotype (V8).
 Contamination of food with Salmonellae especially with S. Typhimurium was high and indicated a bad microbiological quality of food. This emphasizes the need for rigorous public health and food safety methods to lower the human health hazard and risk associated with Salmonellae infection.

Crystal structures of PirA and PirB toxins from Photorhabdus akhurstii subsp. akhurstii K-1

PirAB binary toxin from Photorhabdus is toxic to the larvae of dipteran and lepidopteran insect pests. However, the 3-D structures and their toxicity mechanism are not yet fully understood. Here we report the crystal structures of PirA and PirB proteins from Photorhabdus akhurstii subsp. akhurstii K-1 at 1.6 and 2.1 Å, respectively. PirA comprises of eight β-strands depicting jelly-roll topology while PirB folds into two distinct domains, an N-terminal domain (PirB-N) made up of seven α-helices and a C-terminal domain (PirB-C) consists of ten β-strands. Despite the low sequence identity, PirA adopts similar architecture as domain III and PirB shared similar architecture as domain I/II of the Cry δ-endotoxin of Bacillus thuringiensis, respectively. However, PirA shows significant structural variations as compared to domain III of lepidopteran and dipteran specific Cry toxins (Cry1Aa and Cry11Ba) suggesting its role in virulence among range of insect pests and hence, in receptor binding. High structural resemblance between PirB-N and domain I of Cry toxin raises the possibility that the putative PirAB binary toxin may mimic the toxicity mechanism of the Cry protein, particularly its ability to perform pore formation. The mixture of independently purified PirA and PirB proteins are not toxic to insects. However, PirA-PirB protein complex purified from expression of pir operon with non-coding Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences found toxic to Galleria mellonella larvae with LD50 value of 1.62 μg/larva. This suggests that toxic conformation of PirA and PirB are achieved in-vivo with the help of ERIC sequences.

Polymerase chain reaction-based typing methods and protein profiling analysis of Acinetobacter baumannii isolated from environmental and clinical sources from South India.

Acinetobacter baumannii is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in infected individuals' blood, pus, and respiratory pathways. Due to its resilient nature, it is known to be a causative agent for outbreaks. Therefore, it is crucial to understand the genetic similarity between clinical and environmental isolates. The study aimed to find the genetic relationships between clinical and environmental isolates using PCR-based typing methods such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), random amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic sequence-based PCR (Rep-PCR). Additionally, outer membrane protein (OMP) and whole cell protein (WCP) profiles were also used. The PCR-based methods, ERIC-PCR and Rep-PCR, showed decreased genetic similarity between clinical and environmental isolates (66% and 58%, respectively). However, RAPD showed relatively higher genetic similarity (91%). The OMP and WCP profiles showed varied banding patterns between the clinical and environmental isolates in the 29-43 kDa region. The PCR-based methods proved to be a reliable and reproducible technique. The OMP and WCP profiles, though not as discriminatory as the molecular typing methods, could help identify the most and least commonly occurring protein bands and thus help in typing clinical and environmental A. baumannii isolates.

Repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence‐based typing of Shiga toxin‐producing Escherichia coli from bovine samples

AbstractThe strain typing of Shiga toxin‐producing Escherichia coli (STEC) is challenging due to its high genetic variability. In the present study, two polymerase chain reaction (PCR)‐based typing methods—repetitive extragenic palindromic sequences PCR (REP‐PCR) and enterobacterial repetitive intergenic consensus sequences PCR (ERIC‐PCR)—are evaluated for their efficacy in differentiating the strains. The present study used 100 strains of STEC isolated from bovine meat, bovine feces, and swabs from the chopping boards. The REP‐PCR profiles showed 21 different banding patterns, whereas ERIC‐PCR profiles obtained 18 patterns among the tested strains. The dendrogram prepared by the neighbor‐joining method formed eight and seven clusters for REP and ERIC‐PCR, respectively. The discriminatory indices for the typing methods were 87.83 and 83.03% for REP and ERIC‐PCR, respectively. Thus, the clusters based on REP and ERIC‐PCR fingerprints were unambiguous and thus could be useful for phylogenetic clustering and epidemiological surveillance. The nonparametric multidimensional scaling plots showed more than 75 and 60% similarities within populations of different REP and ERIC clusters, respectively, regarding its source of isolation, serogroup, and distribution virulence markers. The REP and the ERIC sequences are good molecular markers that offer rapid, cost‐effective, and easy molecular typing for STEC strains.

Incidence and Molecular Characterization of Carbapenemase Genes in Association with Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from Tertiary Healthcare Facilities in Southwest Nigeria.

Pseudomonas aeruginosa, resistant to multiple antibacterial agents including carbapenems, is of great global public health concern. There is limited data available regarding incidence of Metallo-Beta Lactamase producing P. aeruginosa, their molecular basis of resistance in particular carbapenem resistance and any genetic relatedness among circulating clinical isolates in Southwest Nigeria. Four hundred and thirty P. aeruginosa isolates were collected from seven tertiary care hospitals (predominantly from wound, ear, and urinary tract infections) and verified by PCR targeting oprI and oprL. Antibiotic susceptibility using 16 selected antibiotics and MBL screening was performed. The integrons (class 1, 2 and 3) and carbapenemase genes- blaGES, blaNMC-A, blaBIC-1, blaSME, blaIMP, blaVIM, blaSPM, blaNDM, blaAIM, blaDIM, blaSIM, blaGIM, blaOXA-48, blaOXA-58 were detected by PCR and were sequenced. Quantitative real-time polymerase chain reaction was used to quantify expression levels of eight efflux pump genes, ampC cephalosporinase and outer membrane porin, oprD. The isolates were genotyped using Enterobacterial Repetitive Intergenic Consensus sequence Polymerase Chain Reaction (ERIC-PCR). Four hundred and thirty P. aeruginosa isolates were subjected to antibiotic susceptibility testing, revealing that 109 (25.4%) isolates were multidrug-resistant, 47 (10.9%) were extensively drug-resistant and 25 (5.8%) were pandrug-resistant. MBL was seen in 17.0% (73/430) isolates. MBL-encoding genes; blaVIM-5 and blaNDM-1 were detected in 86.3% (63/73) isolates, with blaVIM-5 and blaNDM-1 in 35.6% (26/73) and 38.4% (28/73), respectively, whereas co-occurrence of blaVIM-5 and blaNDM-1 was found in 12.3% (9/73). Forty-one (56.2%) carbapenem-resistant P. aeruginosa strains carried class 1 integrons, while co-occurrence of class 1 and 2 integrons was seen in 12.3%. qPCR results indicated that MexXY-OprM was highly expressed pump in 58.9%, ampC upregulated in 26.0%, while oprD porin was downregulated in 65.8% isolates. ERIC-PCR results suggest that carbapenem-resistant strains exhibit genetic heterogeneity. The high incidence of MBL-encoding genes and integrons in diversified clinical P. aeruginosa from southwestern Nigeria is of great concern. The co-occurrence of blaVIM-5 and blaNDM-1 as well as resistance in general manifesting a gradient based on genotypic variation suggests that there is a strong need for efficient surveillance programs and antibiotic stewardship.

Detection of β-Lactamase–Producing Proteus mirabilis Strains of Animal Origin in Andhra Pradesh, India and Their Genetic Diversity

Proteus mirabilis is abundant in soil and water. Although this bacterium is part of the normal human intestinal flora, it can cause serious infections in humans, including complicated urinary tract infections. This pathogen is also commonly associated with multidrug resistance. In the present study, analysis of 1,093 samples from foods of animal origin and animal intestinal samples recovered 232 P. mirabilis isolates identified by PCR assay. Of these 232 isolates, 72 produced β-lactamase (determined by both phenotypic and genotypic methods), with the highest prevalence in poultry cloacal swabs (11.82%) followed by mutton (9.18%), khoa (6.32%), pork (5.63%), pig rectal swabs (5.52%), beef (5.45%), and chicken (5.13%) but none from sheep rectal swabs and bovine rectal swabs. Among β-lactamase genes, blaTEM was the predominant gene detected (59 isolates) followed by blaOXA (11 isolates), blaSHV (5 isolates), blaFOX (5 isolates), blaCIT (4 isolates), blaCTX-M1 and blaCTX-M9 (2 isolates each) and blaCTX-M2, blaDHA, and blaEBC (1 isolate each). None of the isolates carried blaACC, blaMOX, or carbapenemase genes (blaVIM, blaIMP, blaKPC, and blaNDM-1). Dendrogram analysis of enterobacterial repetitive intergenic consensus sequences and repetitive extragenic palindromic sequences obtained with PCR analysis of β-lactamase-producing isolates revealed 63 isolates, but 9 isolates did not yield bands. The analysis revealed that 6.58% of the samples had β-lactamase-producing P. mirabilis isolates that may affect food safety and contaminate the environment. Further genotyping revealed the genetic relationships between isolates of different origin. These findings emphasize the need for careful use of antibiotics to control the spread of β-lactamase-producing bacteria.

Diversity of Vibrio parahaemolyticus in marine fishes of Bangladesh.

To determine the occurrence, diversity, antibiotic resistance and biofilm formation of Vibrio parahaemolyticus isolated from marine fishes in Bangladesh. A total of 80 marine fishes were obtained from the local markets and examined for the presence of V. parahaemolyticus. All the isolated V. parahaemolyticus were characterized for the presence of virulence markers, thermostable direct hemolysin (TDH) or thermostable direct hemolysin related hemolysin (TRH). Isolates were serotyped and further characterized by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing to analyse the genetic diversity. Moreover, biofilm formation and antibiotic resistance patterns were also determined. About 63·75% (51/80) of the tested marine fishes were contaminated with V. parahaemolyticus. From the contaminated fishes, 71 representatives V. parahaemolyticus were isolated and none of them harboured tdh and trh virulence genes. Nine different O-groups and seven different K-types were found by serological analysis and the dominant serotype was O5:KUT. In ERIC-PCR analysis, eight clusters (A-H) were found and the most common pattern was A (46·5%). All of the isolates were resistant to ampicillin and 78·9% of isolates were resistant to streptomycin. The highest biofilm formation was found at 37°C compared to 25°C and 4°C. Diverse V. parahaemolyticus are present in marine fishes in the local market of Bangladesh with antibiotic-resistant properties and biofilm formation capacity. The widespread prevalence of diverse V. parahaemolyticus in marine fishes is an issue of serious concern, and it entails careful monitoring to ascertain the safety of seafood consumers.

Fecal Carriage and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae from Inpatient Children in a Pediatric Hospital of Shanghai.

PurposeTo determine the epidemiology characteristics of intestinal colonization of carbapenem-resistant Enterobacteriaceae (CRE) among inpatients in a pediatric hospital in China.MethodsA retrospective study was conducted from April to December 2019. Medical records were reviewed to extract the clinical information. Antimicrobial susceptibility was performed by broth microdilution method. Drug resistance determinants and plasmid types were analyzed using polymerase chain reaction (PCR) assays. Multilocus sequence typing (MLST) and Enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) were employed to determine the genetic relationships between strains.ResultsA total of 90 CRE strains were isolated, with a fecal carriage rate of 8.6% (90/1052), and mainly distributed in E. aerogenes (n=30), K. pneumoniae (n=25) and E. coli (n=23). More than 50% of CRE colonizers had a history of invasive procedures and antibiotic exposures. As high as 91.1% (82/90) of CRE isolates carried carbapenemase genes, with blaNDM-5 (n=56) being the most common, and mainly found in E. aerogenes (51.8%, 29/56) and E. coli (32.1%, 18/56) isolates, which primarily belonged to ST4 (100%, 29/29) and ST692 (55.6%, 10/18), respectively. Followed by blaKPC-2 (n=12), and all found in K. pneumoniae ST11 isolates. Other carbapenemase genes including blaNDM-1, blaIMP-4 and blaIMP-26. Meanwhile, ESBL genes (blaCTX-M, blaTEM-1 and blaSHV) and AmpC genes (blaDHA-1 and blaEBC) were also detected. All CRE isolates showed high resistance to cephalosporins and carbapenemases (97.8%-100.0%) but remained susceptible to tigecycline (98.9%). IncX3 was a major plasmid type in NDM-containing strains (91.3%), and 91.7% of KPC-2-producing K. pneumoniae harboring IncFII and IncFIB plasmids. The ERIC-PCR revealed that several strains with identical STs were genetically similar.ConclusionThis study revealed a major intestinal colonization of ST4 NDM-5 E. aerogenes, ST11 KPC-2 K. pneumoniae and ST692 NDM-5 E. coli strains among inpatients in a pediatric hospital. Infection control measures should be implemented immediately to prevent the spread of these strains in clinical settings.

First Identification and Characterization of Lactococcus garvieae Isolated from Rainbow Trout (Oncorhynchus mykiss) Cultured in Mexico.

Simple SummaryLactococcus garvieae is an emerging pathogen that causes lactococcosis, which is a septicemia disease that, as per reports, primarily affects rainbow trout and tilapia in various parts of the world. In rainbow trout, the clinical disease is very similar to other diseases caused by Gram-positive bacteria, including weissellosis, which has been previously reported in trout in Mexico. Following septicemia outbreaks that tested negative for weissellosis, a study was conducted to determine the etiology. This study confirms, for the first time, the presence of L. garvieae in processes of a septicemia disease affecting five rainbow trout farms in Mexico. Fourteen isolates were obtained from different geographical regions, but presented homogenous biochemical and protein profiles, as well as weak genetic heterogeneity. This study evidences the wide distribution and incidence of lactococcosis cases at rainbow trout farms in Mexico, demonstrating the need to develop vaccination programs against this disease.Lactococcosis is a hyperacute hemorrhagic septicemia disease caused by Lactococcus garvieae, which is an emerging pathogen in global fish farming. Between 2016 and 2018, rainbow trout (Oncorhynchus mykiss) from five farms that presented outbreaks were sampled as part of a Mexican surveillance program for the detection of fish diseases. Fourteen L. garvieae isolates were recovered from sampled fish, as confirmed by biochemical tests, 16S rRNA gene sequencing, and clinical and histological insights. The biochemical and protein profiles of the isolates obtained were homogeneous. Repetitive extragenic palindromic—(REP)—and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) analyses established weak genetic heterogeneity. Rainbow trout challenged with two of the isolates used at different bacterial concentrations (10−2 and 10−4 CFU/mL) showed melanosis, and hemorrhages were observed in the fins, liver, kidney, and spleen. Isolates were obtained from all of the organs sampled, including from surviving fish, as either pure or mixed cultures. The present study is the first to confirm the presence of L. garvieae as the agent of severe lactococcosis outbreaks in the two primary Mexican states for trout farming.

Prevalence, antibiotic susceptibility and characterization of Vibrio parahaemolyticus isolates in China.

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses a major threat to human health worldwide. In this study, from 2017 to 2019, we evaluated 900 food samples collected from China in 2017, with the aim of determining the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods, shrimp and fish in China. The contamination rates in these were 3.67, 19.33 and 10.67%, respectively, and the prevalence of V. parahaemolyticus was higher in summer than in winter. In addition, 101V. parahaemolyticus strains were isolated. Our results suggested that most of the isolates were resistant to aminoglycosides based on the antimicrobial resistance patterns of these aquatic product isolates against 14 antimicrobial agents. Furthermore, most of the isolates were multidrug-resistant. Serotyping showed that the isolates of the O2 serotype comprised the maximum proportion. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR results indicated that the isolates (n=101) could be classified into 12 clusters. There were 82 STs suggesting genetic variation and relatedness among these isolates. Our findings demonstrated the presence of V. parahaemolyticus in foods from Chinese retail markets and show that this methodology can be used for microbiological risk assessment in China.

Prevalence and antimicrobial resistance of retail-meat-borne Salmonella in southern China during the years 2009–2016: The diversity of contamination and the resistance evolution of multidrug-resistant isolates

Salmonella, one of the most important foodborne pathogens, can be the cause of bacterial food-borne illness and is commonly associated with the consumption of retail meat. Multidrug-resistant Salmonella isolates with high adaptability, have been responsible for many foodborne disease outbreaks. Here we present an investigation on the contamination and the antimicrobial resistance of Salmonella in retail meat obtained from supermarkets and from open markets in Guangxi, China. From the years 2009 to 2016, a total of 604 Salmonella isolates were recovered from a total of 3340 meat samples including 797 beef, 911 pork, 942 chicken and 690 duck, representing 18.08% of the samples tested. Pork was the most contaminated meat. Salmonella was detected in 322 samples from supermarkets and the positive rate of 21.03% was higher than that of 15.70% in 284 samples from open markets (P<0.05). The prevalence of Salmonella in retail meat in the summer and fall months: June (2015, 40.63%), October (2012, 34.6%; 2016, 43.75%) was higher than in other seasons of the year. One hundred and twenty-seven serotypes were identified among the 604 Salmonella enterica isolates, and S. Derby (28.48%), S. Agona (9.77%), S. London (4.97%) and S. Enteritidis (4.47%) were the most common serotypes. Tests of susceptibility to 21 antimicrobial agents showed that 87.58% of the isolates were resistant to at least one antimicrobial, and 57.79% exhibited multidrug resistance (MDR), as they were resistant to at least three antimicrobials. The presence of most of the antimicrobial-resistant genes tested was consistent with the resistant phenotypes found. Among all the antimicrobial resistant genes (ARGs) examined in this study, blaTEM-1, aadA1, cmlA, tetA, sul1 and sul2 were the most prevalent resistant genes in the multidrug resistant isolates. Our findings show that there was a trend that the Salmonella contamination in retail meat had increased and isolates showed an MDR phenotype and that the MDR had become more and more serious. Twenty-one isolates of S. Agona were randomly analyzed by using the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and six different types were found, indicating the existence of cross-contamination in the food market. The results indicate that the hazard analysis of the critical control points (HACCP) system for the whole food chain of retail meat should be further analyzed and improved.

Detecting and molecular typing of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a prevalent food-borne pathogen in many Asian countries and has been spreading rapidly following the appearance of pandemic strains. This article reviews conventional and molecular methods for culturing, identifying, enumerating and rapidly detecting V. parahaemolyticus and its virulence factors, such as thermostable direct hemolysin (TDH), TDH-related hemolysin and thermolabile hemolysin. It also discusses various molecular typing methods, namely, pulsed-field gel electrophoresis, ribotyping, random amplified polymorphic DNA and three other polymerase chain reaction methods based on specific conserved nucleotide sequences, such as ribosomal gene spacer, repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequences, for use in epidemiological investigations of this pathogen.

Application of machine learning algorithm and modified high resolution DNA melting curve analysis for molecular subtyping of Salmonella isolates from various epidemiological backgrounds in northern Thailand.

Food poisoning from consumption of food contaminated with non-typhoidal Salmonella spp. is a global problem. A modified high resolution DNA melting curve analysis (m-HRMa) was introduced to provide effective discrimination among closely related HRM curves of amplicons generated from selected Salmonella genome sequences enabled Salmonella spp. to be classified into discrete clusters. Combination of m-HRMa with serogroup identification (ms-HRMa) helped improve assignment of Salmonella spp. into clusters. In addition, a machine learning (dynamic time warping) algorithm (DTW) was employed to provide a simple and rapid protocol for clustering analysis as well as to create phylogeny tree of Salmonella strains (n = 40) collected from home, farms and slaughter houses in northern Thailand. Applications of DTW and ms-HRMa clustering analyses were capable of generating molecular signatures of the Salmonella isolates, resulting in 25ms-HRM and 28 DTW clusters compared to 14 clusters from a standard HRM analysis, and the combination of both analyses permitted molecular subtyping of each Salmonella isolate. Results from DTW and ms-HRMa cluster analyses were in good agreement with that obtained from enterobacterial repetitive intergenic consensus sequence PCR clustering. While conventional serotyping of Clusters 1 and 2 revealed six different Salmonella serotypes, the majority being S. Weltevraden, the new Salmonella subtyping protocol identified five S. Weltevraden subtypes with S.Weltevreden subtype DTW4-M1 being predominant. Based on knowledge of the sources of Salmonella subtypes, transmission of S. Weltevraden in northern Thailand was likely to be farm-to-farm through contaminated chicken stool. In conclusion, the rapid, robust and specific Salmonella subtyping developed in the study can be performed in a local setting, enabling swift control and preventive measures to be initiated against potential epidemics of salmonellosis.

Molecular characterization of Bacillus thuringiensis strains to control Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) population

Abstract The main objective of this study was to characterize the toxicity and genetic divergence of 18 Bacillus thuringiensis strains in the biological control of Spodoptera eridania. Bacterial suspensions were added to the S. eridania diet. Half of the selected B. thuringiensis strains caused high mortality seven days after infection. The genetic divergence of B. thuringiensis strains was assessed based on Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromic (REP) sequences, and five phylogenetic groups were formed. Despite their genetic diversity B. thuringiensis strains did not show any correlation between the collection sites and toxicity to larvae. Some B. thuringiensis strains are highly toxic to S. eridania, thus highlighting the potential of their endotoxins as biopesticides.

Prevalence, virulence, antimicrobial resistance, and molecular characterization of fluoroquinolone resistance of Vibrio parahaemolyticus from different types of food samples in China

Vibrio parahaemolyticus is the leading cause of foodborne bacterial poisoning in China. The aim of this research is to conduct a study on the prevalence, virulence, and antimicrobial resistance of V. parahaemolyticus from different types of food samples in 12 different cities of China. Since fluoroquinolones are the major choice of treatment for V. parahaemolyticus infections, the genetic basis for fluoroquinolone resistance in V. parahaemolyticus were also investigated. V. parahaemolyticus was detected in 163 of the 784 food samples collected from 12 different cities in China, resulting in a prevalence of 20.79%. The prevalence of V. parahaemolyticus in ready-to-eat (RTE) food (4.96%) was much lower than those of shrimp (32.62%) and fish (22.00%). Virulence gene screening showed that 44 (27.00%) V. parahaemolyticus strains carried at least one virulence gene. Four isolates from shrimp and three isolates from fish contained both the virulence genes tdh and trh. In addition, the trh was firstly detected in one isolate collected from RTE food. All isolates exhibited relatively high resistance rates to ampicillin (82.21%), gentamicin (19.63%), and tetracycline (14.11%), while <10% of strains were resistant to ciprofloxacin (4.91%), levofloxacin (4.91%), and tetracycline (4.29%). Eight fluoroquinolone-resistant V. parahaemolyticus were selected to determine the molecular basis for fluoroquinolone resistance. These eight isolates belonged to three different types according to enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). A Ser83Ile substitution in GyrA was deteted in seven fluoroquinolone-resistant strains, except V209 which harbored a Ser83Phe substitution in GyrA. Moreover, A Ser85Leu substitution in ParC was found in five isolates (V52, V53, V61, V163, and V209). Plasmid-mediated quinolone resistance (PMQR) genes were detected in all eight fluoroquinolone-resistant V. parahaemolyticus strains. This is the first report of Ser83Phe substitution in GyrA, qnrD and qnrS1 in V. parahaemolyticus. The information generated in this study will provide valuable information for risk assessment of V. parahaemolyticus infections and future control of antibiotic-resistant V. parahaemolyticus species in China.

Antibiotic Resistance And Genotyping Of Gram-Positive Bacteria Causing Hospital-Acquired Infection In Patients Referring To Children's Medical Center.

IntroductionA serious problem affecting human society is the development of bacterial resistance. The purpose of the current study was to evaluate the antibiotic resistance of Gram-positive bacteria (GPB) and genotyping of common GPB causing hospital-acquired infections (HAIs) in patients who were referred to Children’s Medical Center during a 6-month period by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR).MethodsDuring the 6-month period, antimicrobial resistance profiles of GPB isolates recovered from patients in Children’s Medical Center were determined using the Kirby–Bauer disk diffusion and MIC. Typing of common GPB was performed and the results were analyzed by gel compare software.ResultsIn this cross-sectional study, 6524 cultures were performed and 138 Ggram-positive bacteria were isolated (2%). Staphylococcus aureus strains showed the highest antibiotic penicillin resistance (96.3%). Twenty-six per cent of the strains were methicillin-resistant S. aureus (MRSA) and no resistance was found against vancomycin. All isolates of Enterococcus faecium were resistant to ciprofloxacin (100%). The resistance to vancomycin was very high (67%) and no resistance was observed to linezolid. The results of genotyping analysis of S. epidermidis strains showed the presence of two clones with a genetic relationship of over 80%. All of the S. aureus strains were in one cluster and half of the E. faecium strains were in a cluster with a genetic predilection of over 80%.ConclusionThis study indicated frequent occurrence of antimicrobial resistance, especially in Enterococcus spp. isolates. Rapid spreads of MRSA and VREF from a clonal origin require implementing careful isolation and infection control measures.

Genetic relatedness in carbapenem-resistant isolates from clinical specimens in Ghana using ERIC-PCR technique.

AimEnterobacterial repetitive intergenic consensus (ERIC) sequence analysis is a powerful tool for epidemiological analysis of bacterial species. This study aimed to determine the genetic relatedness or variability in carbapenem-resistant isolates by species using this technique.MethodsA total of 111 non-duplicated carbapenem-resistant (CR) Gram-negative bacilli isolates from a three-year collection period (2012–2014) were investigated by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC–PCR) in four selected hospital laboratories in Ghana. The isolates were also screened for carbapenemase and extended spectrum β-lactamase genes by PCR.ResultsA proportion of 23.4% (26/111) of the genomic DNA extracts were carriers of PCR-positive carbapenemase genes, including 14.4% blaNDM-1, 7.2% blaVIM-1 and 1.8% blaOXA-48. The highest prevalence of carbapenemase genes was from non-fermenters, Acinetobacter baumannii and Pseudomonas aeruginosa. For the ESBL genes tested, 96.4% (107/111) of the CR isolates co-harboured both TEM-1 and SHV-1 genes. The ERIC-PCR gel analysis exhibited 1 to 8 bands ranging from 50 to 800 bp. Band patterns of 93 complex dissimilarities were visually distinguished from the 111 CR isolates studied, while the remaining 18 showed band similarities in pairs.ConclusionOverall, ERIC-PCR fingerprints have shown a high level of diversity among the species of Gram-negative bacterial pathogens and specimen collection sites in this study. ERIC-PCR optimisation assays may serve as a suitable genotyping tool for the assessment of genetic diversity or close relatedness of isolates that are found in clinical settings.

Enterotoxin gene profile and molecular epidemiology of Aeromonas species from fish and diverse water sources.

This investigation was undertaken to study the prevalence, enterotoxin gene profile and molecular epidemiology of Aeromonads from various sources of water (182) and fish (173). A total of 116 Aeromonas sp. were isolated, of which 48 (26·37%) were from water and 68 (34·62%) were from fish samples collected from retail markets and fish farms. The Aeromonads were recovered from all types of water sources viz. drinking water (13%), surface waters (26%) and fish ponds (69%). The most prevalent species recovered from drinking water was A. hydrophila, from fish ponds it was A. caviae, from surface water sources A. hydrophila and A. caviae were recovered more frequently, and A. hydrophila and A. veronii bv. sobria were isolated predominantly from gills of fish samples. On multiplex PCR analysis for the detection of enterotoxin genes (act, alt, ast), the above mentioned Aeromonas species frequently contained enterotoxin genes, irrespective of their sources. From isolates across all the sources, act (63%) and alt (57%) genes were encountered more frequently than ast (6%). The enterobacterial repetitive intergenic consensus sequences polymerase chain reaction was used for typing of isolates and most of the isolates from water and fish were related, owing to similar ecosystem. A wide distribution of enterotoxin genes in Aeromonads from water and fish is a potential public health threat and molecular genotyping can be helpful to study epidemiology of the pathogen. A high proportion of isolates recovered from diverse water sources, particularly potable drinking water and fish samples carried one or more enterotoxin genes thereby indicating a potential pathogenic nature of isolates from these sources. The genetic relatedness was detected amongst many isolates recovered from water sources and fish samples indicating circulation of familiar virulent clones in the aquatic environments.

Prevalence and genetic diversity of Vibrio parahaemolyticus strains from salmon in Chinese markets.

Consumption of contaminated salmon in China has led to pathogenic Vibrio parahaemolyticus infections in humans. In this study, 420 salmon samples were collected from supermarkets and restaurants in China that showed a contamination rate of 9.05 and 15.24%, respectively. Eighteen antibiotics were used to test the antibiotic susceptibility of all 51 isolates. The most common resistance was observed to β-lactam antibiotics and aminoglycosides, including ampicillin (92.16%), streptomycin (88.24%), kanamycin (45.10%) and cephazolin (45.10%). Meanwhile, many strains were resistant to more than two antibiotics (48/51, 94.12%). Only two and five isolates were positive for tdh and trh, respectively. Serotyping results demonstrated that O2 was most prevalence serotype (15/51, 29.41%). Molecular typing (enterobacterial repetitive intergenic consensus sequence polymerase chain reaction and multilocus sequence typing) allowed classification of all the isolates into 5 clusters and 44 sequence types, highlighting genetic variation and relatedness. In general, the high antibiotic resistance is alarming and raises public health concerns. Frequent monitoring of salmon for V. parahaemolyticus contamination, genetic diversity and antibiotic susceptibility is essential to improve seafood safety.