Plasmids and bacterial strains mediating multidrug-resistant hospital-acquired infections are coresidents of the hospital environment.

Hospital-acquired infections (HAIs) are a global problem. The widespread use of antibiotics continues to exacerbate the problem giving rise to antibiotic-resistant bacteria both in and outside a clinical context. The general hospital environment is an obvious important focus for the selection and spread of multiresistant bacteria and a potential direct source of HAIs. Despite this, there are few detailed studies that have investigated the relationship between strains mediating HAIs and strains coresident in the hospital. Here we isolated bacteria from patients with HAIs exhibiting resistance to ß-lactam antibiotics over a 1-month period in 2011. Three of these isolates were examined in detail by molecular analysis and their multiresistance regions were compared to ß-lactam resistant bacteria isolated from the immediate hospital environment over the same period. All sampled patients were in a 14-bed burns unit and the environmental sample sites included shower drains, sinks, trolleys, and door handles. It was found that identical strains carrying the same resistance regions were present in both patients and the hospital environment suggesting HAIs can arise from bacteria resident in the immediate surrounds. The three patient infections were not derived from a single source, since strains could be distinguished by the genotype and spatial location. While it seems unlikely that eradication of multiresistant bacteria from the hospital can be achieved, more effective hospital cleaning and a better hospital design may be able to reduce transmission.

Genetic context and structural diversity of class 1 integrons from human commensal bacteria in a hospital intensive care unit.

Most surveys for class 1 integrons are at least partly predicated on PCR screening that targets integron conserved regions. However, class 1 integrons are structurally diverse, so dependence on conserved regions may lead to missing clinically relevant examples of class 1 integrons. Here, we surveyed a commensal population of bacteria from patients in an intensive care unit to identify class 1 integrons irrespective of their structure or genetic context. We identified several examples of class 1 integrons linked to complete Tn402-like or Tn402 hybrid transposition modules and diverse insertion points with respect to the inverted repeat IRi boundary. The diversity and abundance of class 1 integrons identified are such that many novel elements seen here would not have been identified by commonly used methods, and they revealed an additional level of complexity.

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli.

BACKGROUND: Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σS (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain. RESULTS: Nine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms. CONCLUSIONS: The ppGpp and σS variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.

Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures.

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA-oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.

Genomic sequencing reveals regulatory mutations and recombinational events in the widely used MC4100 lineage of Escherichia coli K-12.

The genome of an Escherichia coli MC4100 strain with a lambda placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.

Fluorophore labeling to monitor tRNA dynamics.

Transfer RNA (tRNA) molecules mediate translation of the nucleic acid genetic code into the amino acid building blocks of proteins, thus ensuring the survivability of cells. The dynamic properties of tRNA molecules are crucial to their functions in both activity and specificity. This chapter summarizes two methods that have been recently developed or improved upon previous protocols to introduce fluorophores to site-specific positions in tRNA. One method enables incorporation of fluorophores carrying a primary amine (such as proflavin or rhodamine) to dihydrouridine (D) residues in the tRNA tertiary core, and a second method enables incorporation of pyrroloC and 2-aminopurine to positions 75 and 76, respectively, of the CCA sequence at the 3' end. These site-specific fluorophore labeling methods utilize tRNA transcripts as the substrates to provide the versatility with both wild-type and mutant sequences for examining their motions in space and time during the process of decoding genetic information.

Fluorescent labeling of tRNAs for dynamics experiments.

Transfer RNAs (tRNAs) are substrates for complex enzymes, such as aminoacyl-tRNA synthetases and ribosomes, and play an essential role in translation of genetic information into protein sequences. Here we describe a general method for labeling tRNAs with fluorescent dyes, so that the activities and dynamics of the labeled tRNAs can be directly monitored by fluorescence during the ribosomal decoding process. This method makes use of the previously reported fluorescent labeling of natural tRNAs at dihydrouridine (D) positions, but extends the previous method to synthetic tRNAs by preparing tRNA transcripts and introducing D residues into transcripts with the yeast enzyme Dus1p dihydrouridine synthase. Using the unmodified transcript of Escherichia coli tRNAPro as an example, which has U17 and U17a in the D loop, we show that Dus1p catalyzes conversion of one of these Us (mostly U17a) to D, and that the modified tRNA can be labeled with the fluorophores proflavin and rhodamine 110, with overall labeling yields comparable to those obtained with the native yeast tRNAPhe. Further, the transcript of yeast tRNAPhe, modified by Dus1p and labeled with proflavin, translocates on the ribosome at a rate similar to that of the proflavin-labeled native yeast tRNAPhe. These results demonstrate that synthetic tRNA transcripts, which may be designed to contain mutations not found in nature, can be labeled and studied. Such labeled tRNAs should have broad utility in research that involves studies of tRNA maturation, aminoacylation, and tRNA-ribosome interactions.