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 ß-lactamases.

Novel variants of AbaR resistance islands with a common backbone in Acinetobacter baumannii isolates of European clone II.

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.

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.

Tigecycline - how powerful is it in the fight against antibiotic-resistant bacteria?

Tigecycline is a semisynthetic analogue of earlier tetracyclines and represents the first member of a novel class of antimicrobials - glycylcyclines - recently approved for clinical use. It is active against a broad range of gram-negative and gram-positive bacterial species including clinically important multidrug-resistant nosocomial and community-acquired bacterial pathogens. The exact molecular basis of tigecycline action is not clear at present, although similarly to the tetracyclines, it has been shown to inhibit the translation elongation step by binding to the ribosome 30S subunit and preventing aminoacylated tRNAs to accommodate in the ribosomal A site. Importantly, tigecycline overcomes the action of ribosomal protection proteins and is not a substrate for tetracycline efflux pumps of most bacteria - well-known and prevalent cellular mechanisms of microbial tetracycline resistance. The present review summarizes current knowledge on the molecular mechanism of the tigecycline action, antibacterial activity against various bacteria, clinical application, development of resistance to glycylcyclines.

Prevalence of trimethoprim resistance genes in Escherichia coli isolates of human and animal origin in Lithuania.

A total of 456 non-repetitive Escherichia coli isolates from human clinical specimens (urinary, n=134; cervix, vagina and prostate, n=52; blood, pus and wounds, n=45), healthy animals (cattle, n=45; poultry, n=20) and diseased animals (cattle, n=53; swine, n=64; poultry, n=43) obtained in Lithuania during the period 2005-2008 were studied for trimethoprim (TMP) resistance and the prevalence of dfr genes. A TMP resistance rate in the range of 18-26 % respective to the origin was found in clinical isolates, 23-40 % in isolates from diseased animals and 9-20 % in isolates from healthy animals. Of 112 TMP-resistant isolates, 103 carried at least one of the six dfrA genes (dfrA1, dfrA5, dfrA8, dfrA12, dfrA14 and dfrA17) as determined by multiplex PCR and RFLP. The dfrA1 and dfrA17 genes were found most frequently in clinical isolates (17 and 19 isolates, respectively), whilst dfrA1 and dfrA14 genes dominated in isolates of animal origin (25 and 13 isolates, respectively). The dfrA5, dfrA12 and dfrA8 genes were detected at lower frequencies. The association with class 1/class 2 integrons was confirmed for 73-100 % of dfr genes found in most groups of isolates, except for the isolates from diseased swine. In this group, the majority of dfr-positive isolates (67 %, 8/12) carried dfrA8 (6/12) or dfrA14 genes (2/12) that were not associated with integrons. Non-integron location was also confirmed for the remaining dfrA8 genes (six clinical isolates and one isolate from diseased cattle) and for dfrA14 genes (two isolates from diseased cattle and swine each). All cassette-independent dfrA14 genes were found to be located within the strA gene. This study on the prevalence and distribution of TMP resistance genes among E. coli isolates of human and animal origin in Lithuania demonstrates that dfr genes are carried most frequently as gene cassettes within class 1 and/or class 2 integrons. However, TMP resistance in some of the isolates was found to be mediated by non-integron-associated dfrA8 and dfrA14 genes, indicating the existence of alternative sources for the spread of resistance.

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.

Acid response of exponentially growing Escherichia coli K-12.

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.

Transcriptional analysis of the acid-inducible asr gene in enterobacteria.

We show here that transcription of the asr gene in Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae and Enterobacter cloacae is strongly dependent on the acidification level of the growth medium, with maximal induction at pH 4.0-4.5 as determined by Northern hybridization analysis. Previous gene array analyses have also shown that asr is the most acid-induced gene in the E. coli genome. Sequence alignment of the asr promoters from different enterobacterial species identified a highly conserved region located at position -70 to -30 relative to the asr transcriptional start site. By deletion of various segments of this region in the E. coli asr promoter it was shown that sequences upstream from the -40 position were important for induction. Transcription from the E. coli asr promoter was demonstrated to be growth-phase-dependent and to require the alternative sigma factor RpoS (sigma(S)) in stationary phase. Transcription of the asr gene was also found to be subject to negative control by the nucleoid protein H-NS.

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.