Vaida Šeputienė

Molecular Characterization of the Acid-Inducible asr Gene of Escherichia coli and Its Role in Acid Stress Response

Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.

Novel Variants of AbaR Resistance Islands with a Common Backbone in Acinetobacter baumannii Isolates of European Clone II

ABSTRACT In this study, the genetic organization of three novel genomic antibiotic resistance islands (AbaRs) in Acinetobacter baumannii isolates belonging to group of European clone II (EC II) comM integrated sequences of 18-, 21-, and 23-kb resistance islands were determined. These resistance islands carry the backbone of AbaR-type transposon structures, which are composed of the transposition module coding for potential transposition proteins and other genes coding for the intact universal stress protein (uspA), sulfate permease (sul), and proteins of unknown function. The antibiotic resistance genes strA, strB, tetB, and tetR and insertion sequence CR2 element were found to be inserted into the AbaR transposons. GenBank homology searches indicated that they are closely related to the AbaR sequences found integrated in comM in strains of EC II (A. baumannii strains 1656-2 and TCDC-AB0715) and AbaR4 integrated in another location of A. baumannii AB0057 (EC I). All of the AbaRs showed structural similarity to the previously described AbaR4 island and share a 12,008-bp backbone. AbaRs contain Tn1213, Tn2006, and the multiple fragments which could be derived from transposons Tn3, Tn10, Tn21, Tn1000, Tn5393, and Tn6020, the insertion sequences IS26, ISAba1, ISAba14, and ISCR2, and the class 1 integron. Moreover, chromosomal DNA was inserted into distinct regions of the AbaR backbone. Sequence analysis suggested that the AbaR-type transposons have evolved through insertions, deletions, and homologous recombination. AbaR islands, sharing the core structure similar to AbaR4, appeared to be distributed in isolates of EC I and EC II via integration into distinct genomic sites, i.e., pho and comM, respectively.

Spread of carbapenem-resistant Acinetobacter baumannii carrying a plasmid with two genes encoding OXA-72 carbapenemase in Lithuanian hospitals

OBJECTIVES To study the molecular epidemiology of Acinetobacter baumannii isolates from Lithuanian hospitals with an emphasis on the characterization of plasmids and antibiotic-resistance genes and their relationship with European clones (ECs) I and II. METHODS PFGE, PCR analysis of ECs and resistance genes, plasmid replicon typing, DNA transformation and sequencing were employed to characterize A. baumannii. RESULTS Of the 444 isolates studied, 230 (52%) and 202 (45%) belonged to ECI and ECII clones, respectively, and showed clone-specific resistance gene profiles. Five plasmids from 6 to 100 kb in size in different combinations (one to four plasmids) were found in A. baumannii isolates, the combination of 9 + 70 kb plasmids in ECI isolates (60%, 137/230) and an 11 kb plasmid in ECII isolates (52%, 106/202) being the most frequent. GR2 and GR6 replicon groups, alone or in combination, were found, with a prevalence of GR2 + GR6 in ECI isolates of 90% (206/230) and a prevalence of GR2 in ECII isolates of 56% (113/202). The vast majority (95%, 165/174) of carbapenem-resistant A. baumannii ECII isolates carried a novel GR2-type plasmid of 11 kb, designated pAB120, which had two copies of a blaOXA-72 gene, flanked by XerC/XerD-like sites and conferred resistance to carbapenems when introduced into a carbapenem-susceptible A. baumannii strain. CONCLUSIONS The spread of carbapenem-resistant A. baumannii in Lithuanian hospitals is strongly associated with strains belonging to ECII and carrying a GR2 plasmid encoding two blaOXA-72 genes. The genetic environment of pAB120 supports the role of site-specific recombination associated with the acquisition of carbapenem-hydrolysing class D β-lactamases.

Transferable class 1 and 2 integrons in Escherichia coli and Salmonella enterica isolates of human and animal origin in Lithuania.

Antibiotic-resistant Escherichia coli (n = 191) and Salmonella enterica (n = 87) isolates of human and animal origin obtained in Lithuania during 2005-2008 were characterized for the presence and diversity of class 1 and 2 integrons. E. coli isolates were obtained from patients with urinary tract infections (UTIs) (n = 59) and both healthy and diseased farm animals, including poultry (n = 54), swine (n = 35), and cattle (n = 43). Isolates of non-typhoidal S. enterica were recovered from salmonellosis patients (n = 37) and healthy animals, including poultry (n = 31) and swine (n = 19). The presence of integrons, their gene cassette structure, and genome location were investigated by polymerase chain reaction, restriction fragment-length polymorphism, DNA sequencing, Southern blot hybridization, and conjugation experiments. Forty percent of the E. coli and 11% of the S. enterica isolates carried class 1 integrons, whereas class 2 integrons were found in E. coli isolates (9%) only. The incidence of integrons in human UTIs and cattle isolates was most frequent (p < 0.01). A total of 23 different gene cassettes within 15 different variable regions were observed. Seven different integron types, all of them transferable by conjugation, were common for isolates from human infections and for one or more groups of animal isolates. The most prevalent integron types contained arrays dfrA1-aadA1 (36%), dfrA17-aadA5 (23%), and dfrA1-sat1-aadA1 (78%). Two E. coli isolates from humans with UTIs harbored class 1 integron on conjugative plasmid with the novel array type of 4800 bp/dfrA17-aadA5Δ-IS26-ΔintI1-aadB-aadA1-cmlA residing on the Tn21-like transposon. Three S. enterica isolates from swine contained class 1 integron with the newly observed array type of 1800 bp/aadA7-aadA7. Integrons of 10 different types of both classes were located on transferable plasmids in E. coli and S. enterica. Our study demonstrated the existence of a considerable and common pool of transferable integrons in E. coli and S. enterica present in clinical and livestock environment in Lithuania.

An Escherichia coli asr mutant has decreased fitness during colonization in a mouse model

The Escherichia coli asr gene, like its homologues in other enterobacteria, is strongly induced by low external pH. The E. coli asr mutant shows weakened ability to adapt to acidic pH. This suggests that the asr gene product is important for enterobacterial species, both commensal and pathogenic, in overcoming acid stress in the stomach and subsequently colonizing the intestine. We examined the relative fitness of an E. coli asr mutant compared to a wild type, by feeding both strains simultaneously to mice and letting them colonize the intestine. Analysis of the bacteria after passage through the intestine showed up to five orders of magnitude less asr mutant than wild type. Transcomplementation of the asr gene on a plasmid partially restored the number of mutants. Similar competition in liquid media demonstrated that the asr mutant has reduced viability during long-term incubation in rich media, but is as fit as the wild type when bacteria are challenged in minimal medium. Competition carried out under different pH conditions proved that pH of the media was not the main determinant leading to the decreased fitness of the asr mutant. This suggests that the asr gene product is important for adaptation to stress conditions other than acidity, including long periods of starvation.

Detection of methicillin-resistant Staphylococcus aureus using double duplex real-time PCR and dye Syto 9.

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) using real-time polymerase chain reaction (PCR) and dye Syto 9 was developed and evaluated. The assay was based on the two duplex reactions run simultaneously. The detection reaction amplified staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and S. aureus-specific 442-bp DNA (Sa442). The control reaction amplified S. aureus-specific nuclease gene nuc and a marker of methicillin resistance, mecA. The method was evaluated by analyzing 214 clinical S. aureus isolates yielding 98.7 % sensitivity, 100 % specificity, 100 % positive predictive value and 96.6 % negative predictive value for detection of MRSA. The detection limit was determined to be 15–80 genome copies per real-time PCR. It was able to discriminate between MRSA, methicillin resistant coagulase negative staphylococci and methicillin susceptible S. aureus (MSSA) isolates containing only small fragments of the right extremity of the SCCmec (MSSA revertants).