Prevalence and Genetic Diversity of C. Jejuni Isolated from Broilers and their Environment Using flaA-RFLP Typing and MLST Analysis

Campylobacter is highly diverse genetically and also undergoes frequent intraspecific recombination. A major source of campylobacteriosis, which is transmitted to humans is found in poultry. The assessment of the genetic diversity among Campylobacter population is critical to our understanding of the epidemiology. The genetic diversity of Campylobacter jejuni isolates in broilers and their environment were investigated by flaA-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). The study revealed that 92.3% of the examined broiler flocks were contaminated with Campylobacter spp. A total number of 35 different flaA types defined by flaA-RFLP were found in 448 C. jejuni isolates originated from broilers, litter, puddles, zones, anteroom and wild birds. The most dominant flaA type was XXV. MLST defined 20 sequence types (STs) belonging to 10 clonal complexes (CCs). Among all the STs 9 isolates (15%) were consigned to 2 different STs (ST-7413 and ST-4800), which could not be assigned. The most common CCs were ST-21 and ST-179. The ST-21 CC was common in broilers and environment (puddle water and concentric zones) and the ST-179 CC was specific to wild birds, but also was found in puddle water and concentric zones.

The Molecular identification of root nodule bacteria from edible crops of Fabaceae family, Kerala

The root nodule bacteria were isolated from three different leguminous plants belonging to the Fabaceae family such as Cajanus cajan, Lablab purpureus and Vigna unguiculata using YEMA medium and identified. The isolates were submitted to 16S rRNA and PCR–RFLP typing. A representative sample was further submitted to sequence analysis of 16S rRNA. Isolates were assigned to two genera and were related to Rhizobium spp. (75%) and Bacillus spp. (25%) respectively. This study opens the doors to researchers to do specific studies on this root nodule bacteria which are symbiotic with these legume plants in this area for their antibacterial activity, medicine and food application, and sustainable agriculture. Rhizobium and other beneficial microbes may be used as biofertilizers as a substitute for chemical fertilizers.

Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting

The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing ofMycobacterium tuberculosisusing the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing ofM. tuberculosisisolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor.

Genetic characterization ofToxoplasma gondiiisolates from Portugal, Austria and Israel reveals higher genetic variability within the type II lineage

SUMMARYThis study compared genetic diversity ofToxoplasma gondiiisolates from Portugal, Austria and Israel. For this, we genotyped 90T. gondiiisolates (16 from Portugal, 67 from Austria and 7 from Israel) using 10 nested PCR-restriction length polymorphism (RFLP) genetic markers and 15 microsatellite (MS) markers. By PCR-RFLP typing, 7 isolates from Portugal chickens were identified as type II (ToxoDB #1 or #3), 4 were type III (ToxoDB #2) and the remaining 4 isolates have unique genotype pattern were designated as ToxoDB #254. One mouse virulent isolate from a bovine fetus (Bos taurus) in Portugal was type I (ToxoDB #10) at all loci and designated as TgCowPr1. All 67 isolates from Austria and 7 from Israel were type II (ToxoDB #1 or #3). By MS typing, many additional genetic variations were revealed among the type II and type III isolates. Phylogenetic analysis showed that isolates from the same geographical locations tend to cluster together, and there is little overlapping of genotypes among different locations. This study demonstrated that the MS markers can provide higher discriminatory power to reveal association of genotypes with geographical locations. Future studies of the type II strains in Europe by these MS markers will be useful to reveal transmission patterns of the parasite.

The Frequency of Methicillin-Resistant Staphylococcus aureus and Coagulase Gene Polymorphism in Egypt

The current study aimed to use Coagulase gene polymorphism to identify methicillin-resistant Staphylococcus aureus (MRSA) subtypes isolated from nasal carriers in Minia governorate, Egypt, evaluate the efficiency of these methods in discriminating variable strains, and compare these subtypes with antibiotypes. A total of 400 specimens were collected from nasal carriers in Minia governorate, Egypt, between March 2012 and April 2013. Fifty-eight strains (14.5%) were isolated and identified by standard microbiological methods as MRSA. The identified isolates were tested by Coagulase gene RFLP typing. Out of 58 MRSA isolates 15 coa types were classified, and the amplification products showed multiple bands (1, 2, 3, 4, 5, and 8 bands). Coagulase gene PCR-RFLPs exhibited 10 patterns that ranged from 1 to 8 fragments with AluI digestion. Antimicrobial susceptibility testing with a panel of 8 antimicrobial agents showed 6 different antibiotypes. Antibiotype 1 was the most common phenotype with 82.7%. The results have demonstrated that many new variants of the coa gene are present in Minia, Egypt, different from those reported in the previous studies. So surveillance of MRSA should be continued.

Isolation and molecular characterization of Listeria spp. from animals, food and environmental samples

A total of 46 Listeria spp. were isolated from 719 samples (milk, bulk tank swabs, cheese, feed, water, faeces and the environment) collected from 415 cattle and 304 sheep over 12 months (from February 2007 to January 2008). These isolates were identified by conventional and PCR techniques as belonging to L. monocytogenes (17.4%), to Listeria innocua (39.1%), to Listeria seeligeri 17.4%), to Listeria grayi (15.2%) and to Listeria welshimeri (11%). No Listeria ivanovii were isolated from any of the samples. Listeria spp. were not isolated from cheese and bulk tank swabs. With regard to seasonal variations most Listeria spp. were isolated in the spring and winter seasons. The eight L. monocytogenes isolates were characterized by PCR-RFLP with AluI and Tsp509I. RFLP typing of the isolates revealed two different profiles with both restriction enzymes. Four and six different profiles were produced in the examination of L. monocytogenes isolates with RAPD analysis with HLWL74 and HLWL85 primers, respectively. This is the first report on the genotyping of L. monocytogenes isolates from various sources in Turkey. This study has highlighted the need for improved control and epidemiologic strategies to prevent the transmission of Listeria spp. to animals and humans.