From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing, both in human and veterinary medicine. Although initially designed to the treatment of Grampositive microorganisms, this antimicrobial family has also been used to treat specific Gram-negative bacteria. Some of them, as azithromycin, are considered in the armamentarium against Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms of macrolide resistance, currently described in Enterobacteriaceae.

No description available.

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.

There is strong crystallographic evidence that the 23S rRNA is the only catalytic entity in the peptidyl transferase center. Various mechanisms for the catalysis of peptidyl transfer have been proposed. Recently, attention has been given to the idea that the 23S rRNA simply acts to position the tRNA for spontaneous peptidyl transfer and that chemical catalysis may play only a secondary role. Conserved nucleotides U2585 and U2506 are thought to be involved in positioning the 3’ ends of A- and P-site substrates based on the crystallographic evidence, and because mutagenesis at these sites severely impairs peptide bond formation. In this study, pure populations of ribosomes with either U2585A or U2506G mutations in the 23S rRNA were analyzed to test the hypothesis that substitutions at nucleotides U2585 and U2506 in the peptidyl transferase center impair peptide bond formation by altering the position of the 3’ end of P-site tRNA relative to the 23S rRNA. Pure populations of mutant or wild-type ribosomes were obtained by an affinity tagging system and probed with 32P-labeled [2N3A76]tRNAPhe to determine how the 3’ end of tRNA interacts with the ribosomal proteins and 23S RNA at the peptidyl transferase center. Some of the data for the ribosomes with a G at position 2506 are consistent with a model suggested by Schmeing and coworkers in which nucleotide U2506 breaks from its original wobble base pair with nucleotide G2583 during A-site tRNA binding and swings towards the 3’ end of P-site tRNA, while nucleotide U2585 simultaneously moves away from the 3’ end of P-site tRNA.

Abstract The aim of this work was to assess and improve the enrichment cultivation of food-contaminating bacteria prior to detection by means of RNA-based sandwich hybridization assay (SHA). The examples of beer-spoiling lactic acid bacteria (LAB) and food-borne Salmonella Typhimurium were selected based on their relevance in Finnish food industry. Also universal challenges affecting on the selection of the enrichment cultivation procedure are discussed, including some potential possibilities for improved enrichment cultivation. The results of this study may therefore be used for the assessment of the efficiency of bacterial cultivation in other applications. The evaluation of the enrichment cultivation procedures prior to SHA lead to following conclusions: i) the enrichment cultivation procedure is necessary prior to rRNA-based SHA, and it directly influences the accuracy of SHA; ii) the improvement of the enrichment cultivation may allow faster recovery and growth of bacteria; iii) the improved recovery of bacteria can be achieved by reducing environmental stress factors in the enrichment culture; and iv) the growth of bacteria may be accelerated by assuring the selectivity of medium and allowing accessibility to growth factors. Several growth factors were studied by means of full factorial design and response surface modeling. Measured cell densities, as well as predicted lag-times and maximum growth rates in the bacterial cultures were used as responses. The results show that small shifts in the cultivation conditions extend the lag-time and decrease the growth rate of both LAB and Salmonella. Besides adjusting the temperature and pH, the growth of LAB was facilitated by reducing osmotic and oxidative stresses in the enrichment medium. In this study, a novel enzyme controlled glucose delivery system was used for the first time in the enrichment cultivation of food-contaminating bacteria. The glucose delivery system improved the growth of LAB in single strain cultures and in actual brewing process samples. The recovery of injured Salmonella was also enhanced by using the glucose delivery system together with selective siderophore ferrioxamine E, both in terms of reduced lag-times and increased growth rates. Based on the SHA, the adjusted BPW broth enhanced the molecular detection of heat-injured Salmonella in meat.

Flera olika arter av mjölksyrabakterier som ingår i släktena Lactobacillus och Bifidobacterium har hittats hos bin och i deras honung. Idag finns ingen effektiv metod för identifiering av bakterierna. Syftet med detta projekt är att utveckla en metod för snabb identifiering genom att hitta lämpliga primers till olika mjölksyrabakterier och därmed få fram en Polymeraskedjereaktion (PCR) metod. Ribosomal ribonukleinsyra (rRNA) generna eller 16S-23S rRNA intergenic spacer region (ISR) används ofta vid design av primers, som därefter används i PCR för att identifiera olika bakterier. Deoxiribonukleinsyra (DNA) visualiseras i agarosgelen med hjälp av SYBRgreen I som fluorescens på ultraviolett (UV)-ljusbord. I detta projekt har 16S rRNA och 16S-23S rRNA ISR amplifierats i enkel PCR och multiplex PCR och visualiserats i agarosgel i försök att identifiera mjölksyrabakterierna. 16S rRNA har visat sig ha mycket liten variation mellan bakterierna och ansågs därför inte lämplig att använda för identifiering av närbesläktade arter. 16S-23S rRNA ISR visade större variation, fram för allt mellan lactobacillerna och bifidobakterierna. Gruppering av bakterierna med hjälp av multiplex PCR gjordes med viss framgång, med undantag av några bakterier som inte hamnade i den förväntade gruppen. Dock behövs fler försök för att stödja dessa resultat. / Several different lactic acid bacterium (LAB) species from the genera Lactobacillus and Bifidobacterium was discovered in bees and in their honey. Today there is no rapid and reliable method to identify these LAB. Therefore a rapid polymerase chain reaction (PCR) method to identify the LAB is needed. The aim of this project is to find primers suitable for the different LAB. Ribosomal ribonucleic acid (rRNA) genes or 16S-23S rRNA intergenic spacer region (ISR) are often used to designing of primers followed by PCR assays, for identification of different bacteria. To visualize deoxyribonucleic acid (DNA) in agarose gels, SYBRgreen I was used as fluorescence and then viewed under ultraviolet (UV) light. In this project the 16S rRNA and 16S-23S rRNA ISR was used as a target in a PCR and a multiplex PCR amplification. The PCR product was analyzed in agarose gel in an attempt to identify the LAB. 16S rRNA sequence have to little variation and is not suitable to identify closely related species. 16S-23S rRNA ISR sequence exhibits greater variations, especially between Lactobacillus and Bifidobacterium. Differentiation of the bacteria into groups by multiplex PCR was done with good result, except for some of the bacteria that did not end up in the expected group. More studys is needed to support these results.

Hansson, L. Detektion av makrolidresistens hos Mycoplasma genitalium med Panther Fusion. Examensarbete i biomedicinsk laboratorievetenskal 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk Vetenskap, 2023.   Mycoplasma genitalium är en sexuellt överförbar mikroorganism som infekterar både män och kvinnor, som behandlas oftast med azitromycin med ett ökande problem av antibiotikaresistens. För M. genitalium är makrolidresistens det främsta hotet mot behandling, och har kopplats till fyra punktmutationer i region V i 23S rRNA-genen: A2071G, A2072G, A2072C samt A2071T (M. genitalium G-37, GenBank NR_077054.1). Projektet har undersökt möjligheten att ersätta nuvarande in house realtids-PCR metod för makrolidresistensbestämning med ett integrerat nukleinsyra-reningssteg och realtids-PCR med Panther Fusion (Hologic) hos Klinisk mikrobiologi i Lund. Under projektet analyserades 55 patientprover som samlades under perioden januari-februari 2023 i Region Skåne, som blivit positiva vid M. genitalium testning. Dessa prover har därefter analyserats av personal med nuvarande ABI-metod för resistensbestämning och sedan analyserats på Panther Fusion. Nuvarande ABI-metod resulterade i positiv signal för 91% (50/55) av patientprover positiva vid M. genitalium analys och makrolidresistensmutation hos 25 % (14/55), medan Panther Fusion metoden resulterade i positiv signal för 81 % (45/55) av positiva M. genitalium prover och påvisade resistensmutation hos 20 % (11/55) av proverna.

CSIR / Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.