Alvydas Pavilonis

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.

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

Antibiotic-resistant Escherichia coli (nu2009=u2009191) and Salmonella enterica (nu2009=u200987) 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) (nu2009=u200959) and both healthy and diseased farm animals, including poultry (nu2009=u200954), swine (nu2009=u200935), and cattle (nu2009=u200943). Isolates of non-typhoidal S. enterica were recovered from salmonellosis patients (nu2009=u200937) and healthy animals, including poultry (nu2009=u200931) and swine (nu2009=u200919). 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 (pu2009<u20090.01). 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.

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).