Repetitive Intergenic Consensus Sequence-based PCR Research Articles

Antimicrobial resistance profiling and molecular typing of ruminant-borne isolates of Clostridium perfringens from Xinjiang, China

ObjectivesClostridium perfringens (C. perfringens) can cause intestinal diseases in livestock and humans, which seriously threatens the healthy development of animal husbandry and human food safety. Here, the characteristics of antimicrobial resistance and molecular typing of ruminant-borne strains of C. perfringens in Xinjiang, China were explored and profiled. MethodsA total of 307 clinical feces collected from ruminants (cattle and sheep) with diarrheal symptoms were screened for C. perfringens. The recovered isolates were characterized in respect to their antimicrobial resistance pattern and molecular typing. ResultsA total of 109 isolates of C. perfringens were isolated from 307 clinical feces of ruminants, most of which displayed the multidrug resistance (MDR) phenotype. Demonstration of the quinolone-resistance gene was the highest among the isolates (70.6%). The multiplex PCR typing based on toxin genes showed that type A and type D strains made up 82.6% (90/109) and 17.4% (19/109), among which, the isolates carrying β2 gene occupied 43.3% (39/90) of type A strains and 31.6% (6/19) of type D strains. These isolates were divided into 6 genotypes (I-VI) by enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) method. A total of 33 ST types (ST1-ST33) were identified by multilocus sequence typing (MLST) method. ConclusionC. perfringens isolates with multidrug resistance (MDR) were frequent and circulating in ruminants. Among them, type A-Ⅰ-ST19 was the dominant genotype of C. perfringens, displaying obvious genetic diversity. This study provided important epidemiological data for the risk assessment of food safety associated with ruminant-borne C. perfringens in Xinjiang, China.

First description of methyltransferases in extensively drug-resistant Klebsiella pneumoniae isolates from Saudi Arabia.

The resistance determinants for carbapenems, fluoroquinolones and aminoglycosides were characterized in 16 extensively drug-resistant Klebsiella pneumoniae (XDRKPN) strains collected from Saudi hospitals during 2014. PCR and sequencing were used to detect: blaKPC, blaNDM, blaVIM, blaIMP-1,blaOXA-48, blaCTX-M, blaTEM, blaSHV and ampC for β-lactam resistance; qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA and mutations of gyrA and parC for fluoroquinolone resistance; and aacA4, aacC2, aadA1, aphA6, armA and rmtB for aminoglycoside resistance. Enterobacterial repetitive intergenic consensus sequence-based PCR was performed to detect the clonal relatedness. All isolates encoded blaCTX-M, aacC2 and aphA6, together with mutations in gyrA and parC. blaOXA-48, blaNDM-1, aadA1, aacA4, qnrB, aac(6')-Ib-cr, armA and/or rmtB were detected in different strains. At least 93.2 % clonal relatedness was detected among these strains. To our knowledge, this is the first report describing XDRKPN encoding at least seven resistance determinants and harbouring methyltransferases in Saudi Arabia.

Investigation into a community outbreak of Salmonella Typhi in Bengaluru, India.

Background & objectives:Outbreaks of infection due to Salmonella enterica servovar Typhi (S. Typhi) are a great threat to public health. A rapid molecular typing method to characterize strains implicated in an outbreak is critical in implementing appropriate control measures. This study was done to demonstrate the power of a PCR-based method to provide rapid insights into the genetic relatedness amongst the Salmonella isolates implicated in a suspected typhoid fever outbreak.Methods:Forty two S. Typhi isolates originating from three geographically distinct areas, with one area suspected to have a single-source outbreak were included in the study. The genetic fingerprint of all isolates was generated using enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR). The antimicrobial susceptibility profiles were also evaluated.Results:ERIC-PCR was found to be rapid and reproducible with a discriminatory index of 0.766. The dendrogram constructed based on ERIC-PCR fingerprinting revealed the existence of 12 distinct genotypes. The location suspected to have an outbreak displayed two genotypes amongst the 24 isolates. The other two locations (18 isolates) displayed genetic heterogeneity. The clonality of the outbreak isolates from the time-matched control isolates was established. The observed antimicrobial susceptibility profiles did not have any discriminatory power to subtype the isolates compared to the genetic fingerprints.Interpretation & conclusions:Our study demonstrated the discriminatory power and value of ERIC-PCR in the typing of S. Typhi isolates and providing valuable epidemiological insights.

Rates of gastrointestinal tract colonization of carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa in hospitals in Saudi Arabia.

Carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPAE) are globally a major medical issue, especially in intensive care units. The digestive tract is the main reservoir for these isolates; therefore, rectal swab surveillance is highly recommended. The purpose of this study was to detect the prevalence of gastrointestinal tract colonization of CRE and CRPAE in patients admitted to intensive care units in Saudi Arabia. This project also aimed to characterize carbapenem-hydrolyzing enzyme production in these isolates. From February to May 2015, 200 rectal swab specimens were screened by CHROMagar KPC. Organism identification and susceptibility testing were performed using the Vitek 2 system. One CRE and 13 CRPAE strains were identified, for a prevalence of 0.5% (1/200) and 6.5% (13/200) respectively. Strains showed high genetic diversity using enterobacterial repetitive intergenic consensus sequence-based PCR. NDM type and VIM type were detected by PCR in four and one CRPAE isolates respectively. ampC overexpression was detected in eight CRPAE isolates using Mueller-Hinton agar containing 1000 μg/mL cloxacillin. CTX-M-15 type was detected in 1 CRE by PCR. The prevalence of CRE strain colonization was lower than that of CRPAE isolates. The detection of NDM and VIM in the colonizing CRPAE strains is a major infection control concern. To our knowledge, this is the first study in Saudi Arabia and the gulf region focusing on digestive tract colonization of CRE and CRPAE organisms and characterizing the mechanisms of carbapenem resistance.

Characterization of Metal Tolerant Serratia spp. Isolates from Sediments of Uranium Ore Deposit of Domiasiat in Northeast India

Three metal tolerant Serratia spp. isolates from sediments of pre-mined uranium ore deposit of Domiasiat, Meghalaya, India were characterized using morphological, biochemical and molecular methods. 16S rRNA gene analysis of the isolates identified one of the isolates (DB-10) as Serratia nematodiphila with 99.33 % pairwise similarity whereas remaining two isolates (DB-11 and DB-15) showed similarity with Serratia marcescens subsp. sakuensis with similarity of 98.78 and 99.54 % respectively. Randomly amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) studies of the isolates revealed genetic diversity among them. The isolates were found to tolerate all the five tested metals namely, uranium, cadmium, copper, zinc and lead and also could resist nine commonly used antibiotics namely, ampicillin, kanamycin, chloramphenicol, erythromycin, gentamicin, aztreonam, tetracycline, imipenem and ciprofloxin. All the three isolates were found to be superior in tolerance and resistance to the reference strain S. marcescens ATCC13880. The isolates showed high efficiency of uranium (VI) removal ranging from 90 to 92 % (21.4–21.9 mg/L) and from 65 to 70 % (309.4–333.2 mg/L) when challenged with 100 μM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions respectively within 1 h of incubation.

Abstract 4872: Gynostemma pentaphyllum saponins alter the gut microflora composition and convert dysbiosis to eubiosis state in xenograft nude mice.

Abstract Increasing evidence has shown that the gut microflora may have a crucial role in many diseases, including cancers. In our previous studies, we have demonstrated that treatment with total saponins of Gynostemma pentaphyllum (GpS) exerts anti-cancer effects in cellular and animal models. Our study showed that an unnatural shift in the microflora composition was observed in nude mice with xenografted tumors induced by GFP/Ras cells. To investigate the potential association between the anti-cancer effect of GpS and the gut microflora, we first investigated whether GpS treatment would affect the microflora composition. Enterobacterial Repetitive Intergenic Consensus sequence-based PCR (ERIC-PCR) was used to analyze the bacterial genomic DNA from the fecal samples of the experimental mice. ERIC-PCR analysis revealed that gut microbiota communities were able to restore back to the non-tumor state (day 0)) after treatment with GpS for 10 days. In addition, we found that GpS treatment can increase the levels of certain beneficial bacteria, including Bifidobacterium spp. and Lactobacillus spp. Furthermore, we defined the detailed changes in the gut microbiome by pyrosequencing of the 16S rRNA gene sequences. We found that Bacteroidetes/Firmicutes ratio showed an increased trend upon GpS treatment in xenograft nude mice. Furthermore, GpS altered certain species of bacteria, including the Clostridium cocleatum and Bacteroides acidifaciens, which have well-documented beneficial effects. In conclusion, this study has demonstrated GpS can potentially regulate and balance the gut microbial ecosystem by increasing symbionts. However, further investigation is required on whether this alteration of the gut microflora is the cause or consequence of the drug action. [This study was partially supported by FRG2/11-12/102 to WLWHsiao] Citation Format: Lei Chen, William C.S. Tai, Frederick CC Leung, Manreet Singh, W.L. Wendy Hsiao. Gynostemma pentaphyllum saponins alter the gut microflora composition and convert dysbiosis to eubiosis state in xenograft nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4872. doi:10.1158/1538-7445.AM2013-4872

Lactobacillus Strain Ecology and Persistence within Broiler Chickens Fed Different Diets: Identification of Persistent Strains

Lactobacilli are autochthonous residents in the chicken gastrointestinal tract, where they may potentially be used as probiotics, competitive exclusion agents, or delivery vehicles. The aim of this study was to use an in vivo model to investigate the effect of diet and competing lactic acid bacteria on the colonization of inoculated Lactobacillus strains, with the goal of identifying strains which can consistently colonize or persist for an extended period of several weeks. Chicken-derived Lactobacillus strains were genetically marked with rifampin resistance and administered on day 0 to chickens fed either a normal commercial diet or a specially formulated high-protein diet. Chickens fed the high-protein diet were also coinoculated with two different mixes of additional lactic acid bacteria. Enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) was used to identify rifampin-resistant isolates recovered from chickens. Three strains, belonging to the species Lactobacillus agilis, Lactobacillus crispatus, and Lactobacillus vaginalis, were commonly reisolated from the chickens on both diets at days 21 and 42. The ability of these strains to persist was confirmed in a second chicken trial. All three strains persisted throughout the production period in the chickens fed a commercial diet, while only the L. agilis and L. vaginalis strains persisted in the chickens fed the high-protein diet. In both in vivo trials, competing lactic acid bacteria modified representation of the strains recovered, with all three stains capable of competing in the presence of one or both mixes of coinoculated strains. The in vivo model successfully identified three persistent strains that will be characterized further.

Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic

Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.

DNA Fingerprinting Analysis of Breakthrough Outbreaks in Vaccine-Protected Poultry Stocks

We report recurrent outbreaks of Yersinia pseudotuberculosis conjunctivitis in ducks and of fowl cholera in geese, occurring in stocks previously vaccinated with inactivated autogenous vaccines. Enterobacterial repetitive intergenic consensus sequence-based PCR and pulsed-field gel electrophoresis indicated reinfection with a new Y. pseudotuberculosis strain and vaccine evasion by the same Pasteurella multocida strain.

Comparative study of three different DNA fingerprint techniques for molecular typing of Shigella flexneri strains isolated in Romania.

In this study, 97 epidemiologically unrelated Shigella flexneri strains isolated during 1994 (69 isolates) and 1997 (28 isolates) were characterised by ribotyping, enterobacterial repetitive intergenic consensus sequence-based PCR typing, and pulsed-field gel electrophoresis. Number of strains belonging to each of the six serotypes is selected equal to their distribution in Romania. The isolates comprise 24 ribotypes based on combination of two restriction patterns obtained with HindlII and PstI, respectively, 7 enterobacterial repetitive intergenic consensus (ERIC)-PCR types, and 92 XbaI pulsed-field gel electrophoresis (PFGE) patterns grouped in 31 pulsotypes at Dice coefficients of 85% similarity. We find no significant difference in the distribution of isolates collected during the two periods. Macrorestriction analysis by PFGE offers maximal discrimination. There seems to be little genetic variability among circulating S. flexneri strains of serotype 2a, suggesting that even a combination of several molecular techniques, including PFGE, could not easily differentiate an outbreak strain from temporally associated independent isolates.