We report the genome sequence of Klebsiella pneumoniae subsp. pneumoniae Ecl8, a spontaneous streptomycin-resistant mutant of strain ECL4, derived from NCIB 418. K. pneumoniae Ecl8 has been shown to be genetically tractable for targeted gene deletion strategies and so provides a platform for in-depth analyses of this species.

Background

Bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.

Results

A small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).

Conclusions

Small RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.

Tigecycline resistance in Klebsiella pneumoniae results from ramA upregulation that causes the overexpression of the efflux pump, AcrAB-TolC. Tigecycline mutants, derived from Ecl8ΔramA, can exhibit a multidrug resistance phenotype due to increased transcription of the marA, rarA, acrAB, and oqxAB genes. These findings support the idea that tigecycline or multidrug resistance in K. pneumoniae, first, is not solely dependent on the ramA gene, and second, can arise via alternative regulatory pathways in K. pneumoniae.

Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

The mechanism of stepwise acquired multidrug resistance in Acinetobacter baumannii isolates from a hospitalized patient was investigated. Thirteen consecutive multidrug-resistant isolates were recovered from the same patient over a 2-month period. The Vitek 2 system identified the isolates as meropenem-sensitive Acinetobacter lwoffii; however, molecular identification showed that the isolates were A. baumannii. Etest revealed that the isolates were meropenem resistant. The presence of oxacillinase (OXA)-type enzymes were investigated by sequencing. The clonal relatedness of isolates was assessed by pulsed-field gel electrophoresis (PFGE). Expression of the genes encoding the efflux pumps AdeB and AdeJ was performed by semiquantitative real-time reverse transcription-PCR (qRT-PCR). The adeRS two-component system was sequenced. All isolates had identical PFGE fingerprints, suggesting clonal identity. The first six isolates were positive for the novel blaOXA-164 gene. The following seven isolates, recovered after treatment with a combination of meropenem, amikacin, ciprofloxacin, and co-trimoxazole showed an increase of >7-fold in adeB mRNA transcripts and a missense mutation in blaOXA-164, converting it to blaOXA-58. Sequencing revealed a novel mutation in adeR. These data illustrate how A. baumannii can adapt during antimicrobial therapy, leading to increased antimicrobial resistance.

This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O-island 51, a 14.93 kb cryptic prophage (CP-933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O-island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE-encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression.