Capture of a novel, antibiotic resistance encoding, mobile genetic element from Escherichia coli using a new entrapment vector.

AIMS: Antimicrobial resistance genes (ARGs) are often associated with mobile genetic elements (MGEs), which facilitate their movement within and between bacterial populations. Detection of mobility is therefore important to understand the dynamics of MGE dissemination and their associated genes, especially in resistant clinical isolates that often have multiple ARGs associated with MGEs. Therefore, this study aimed to develop an entrapment vector to capture active MGEs and ARGs in clinical isolates of Escherichia coli. METHODS AND RESULTS: We engineered an entrapment vector, called pBACpAK, to capture MGEs in clinical E. coli isolates. It contains a cI-tetA positive selection cartridge in which the cI gene encodes a repressor that inhibits the expression of tetA. Therefore, any disruption of cI, for example, by insertion of a MGE, will allow tetA to be expressed and result in a selectable tetracycline-resistant phenotype. The pBACpAK was introduced into clinical E. coli isolates and grown on tetracycline-containing agar to select for clones with the insertion of MGEs into the entrapment vector. Several insertion sequences were detected within pBACpAK, including IS26, IS903B and ISSbo1. A novel translocatable unit (TU), containing IS26 and dfrA8 was also captured, and dfrA8 was shown to confer trimethoprim resistance when it was cloned into E. coli DH5α. CONCLUSIONS: The entrapment vector, pBACpAK was developed and shown to be able to capture MGEs and their associated ARGs from clinical E. coli isolates. We have captured, for the first time, a TU encoding antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that a TU and associated resistance gene has been captured from clinical E. coli isolates using an entrapment vector. The pBACpAK has the potential to be used not only as a tool to capture MGEs in clinical E. coli isolates, but also to study dynamics, frequency and potentiators of mobility for MGEs.

Caries experience and quantification of Streptococcus mutans and Streptococcus sobrinus in saliva of Sudanese schoolchildren.

Streptococcus mutans and Streptococcus sobrinus are among the most commonly isolated bacterial species implicated as etiological agents of dental caries. Details of the composition of the oral microflora related to dental caries should aid in assessing the prevalence and risk of disease at an individual level. The aim of the present study was to determine the presence and relative amounts of S. mutans and S. sobrinus in the saliva samples obtained from schoolchildren in Khartoum State, the Sudan, and to study the association of the amounts of S. mutans and S. sobrinus with caries experience, socioeconomic status and sugar-sweetened snacks in this population. 140 samples, 30 of which were from individuals with caries experience, were examined using quantitative real-time polymerase chain reaction (qRT-PCR) with specific oligonucleotide primers. The mean ratio of fold differences of S. mutans to S. sobrinus was 0.77 (SD 5.4) and 2.29 (SD 6.0) for samples obtained from caries-free and caries-active individuals, respectively. This suggested that the proportion of S. sobrinus was higher than S.mutans in the caries-active group when compared to the caries-free group. An association was found between children with caries-active lesions and the frequent consumption of sticky desserts and higher socioeconomic status. S. sobrinus seems to be associated with caries experience in the studied population. A proposal of caries screening programs designed to test for S. sobrinus in this population may be developed.

Proteomic analysis of ampicillin-resistant oral Fusobacterium nucleatum.

INTRODUCTION: Fusobacterium nucleatum represents one of the predominant anaerobic species in the oral microbiota. Penicillin-resistant F. nucleatum have been isolated from intra- and extraoral infections. This study aimed to assess ampicillin resistance in F. nucleatum by investigating the synthesis of resistance-associated proteins. METHODS: Ampicillin-resistant and ampicillin-susceptible F. nucleatum isolates were obtained from 22 dental plaque samples. Two-dimensional gel electrophoresis and mass spectrometry were used to investigate bacterial protein synthesis. Proteins exhibiting statistically significant quantitative changes between sensitive and resistant isolates were identified using peptide mass mapping and matrix-assisted laser desorption/ionization - time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry. RESULTS: Twenty-three F. nucleatum isolates were recovered from plaque samples and their ampicillin minimum inhibitory concentrations ranged between 0.125 microg/ml and 256 microg/ml. Analysis of the bacterial cellular proteins by two-dimensional gel electrophoresis resolved 154-246 distinct protein spots (mean 212, n = 9). Between 32% and 83% of the protein spots were common for the F. nucleatum isolates. Comparisons of the protein profiles of sensitive and resistant isolates revealed the presence of a 29 kDa protein and significant increases in the synthesis of two proteins at 37 and 46 kDa in the ampicillin-resistant F. nucleatum isolates. These proteins were identified as a class D beta-lactamase, ATP-binding cassette (ABC) transporter ATP-binding protein and enolase, respectively. CONCLUSION: Synthesis of a class D beta-lactamase by ampicillin-resistant F. nucleatum isolates could complicate antimicrobial treatment because these enzymes might confer resistance to many classes of beta-lactam antibiotics. The differences observed in protein synthesis between ampicillin-resistant and ampicillin-susceptible F. nucleatum may contribute to the antibiotic resistance and virulence of these bacteria.

Prevalence of subgingival bacteria resistant to aminopenicillins and metronidazole in dental patients from Yemen and Norway.

The purpose of this study was to assess the prevalence of resistance to aminopenicillins and metronidazole among selected subgingival species in dental patients from Yemen and Norway. Three subgingival samples were collected by paper points from each of 34 Yemeni and 21 Norwegian adult volunteers and then pooled. Each of the 55 pooled samples was plated on fastidious anaerobic blood agar containing 2 microg/mL ampicillin or metronidazole, or no antimicrobial. Species identification of growth was done using DNA-DNA checkerboard hybridisation. The overall proportion of ampicillin resistance among the 18 identified species was 28.9% and 7.9% in the Yemeni and Norwegian samples, respectively, whereas for metronidazole it was 60.3% and 11.3%. The number of species resistant to ampicillin and metronidazole was significantly higher (P < 0.016 and P = 0.0000, respectively) in the Yemeni than in the Norwegian samples.