Emergence and maintenance of multidrug-resistant Escherichia coli of canine origin harbouring a blaCMY-2-IncI1/ST65 plasmid and topoisomerase mutations.

OBJECTIVES: To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. METHODS: MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. RESULTS: Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. CONCLUSIONS: The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

Accumulation of carbapenem resistance mechanisms in VIM-2-producing Pseudomonas aeruginosa under selective pressure.

Pseudomonas aeruginosa has the potential to achieve resistance to carbapenems via the acquisition of carbapenemase-encoding genes, the downregulation of the OprD porin, the overexpression of efflux systems and the overproduction of cephalosporinases. One hundred and fifty carbapenem-non-susceptible isolates from 2008 to 2010 were screened for carbapenemase production, OprD porin loss, efflux pumps overexpression and inducible AmpC beta-lactamase production. For comparison reasons, the presence of the same mechanisms was also assessed in a previous collection of 30 carbapenem-non-susceptible P. aeruginosa isolated between 2003 and 2005. Results showed the accumulation of various resistance mechanisms among VIM-2 producers isolated between 2008 and 2010 with a parallel considerable increase in imipenem MIC90 and the geometric mean of the MIC values of imipenem and meropenem between the two study groups. The accumulation of carbapenem resistance mechanisms highlights the potential of this formidable pathogen for evolutionary success under antibiotic selective pressure.

Hepatitis B reactivation in a renal transplant patient due to a surface antigen mutant strain: a case report.

INTRODUCTION: Reactivation of hepatitis B virus (HBV) is a complication of immunosuppressive treatment in patients with a history of HBV exposure. CASE PRESENTATION: Herein we have reported a case of reactivation after renal transplantation in a 52-year-old male chronic HBV carrier who was treated with hepatitis B immunoglobulin (HBIg) prophylaxis immediately after transplantation in addition to cyclosporine, mycophenolate mofetil and prednisolone for maintenance immunosuppression. After application of rituximab, the patient developed clinical hepatitis with a high load of HBV DNA. Sequence analysis of the surface (S) antigen corresponding to the amino acid residues 101-186 (including the a-determinant region) revealed a genotype D mutant strain, subtype ayw3 with a single amino acid substitution D144E within the S gene. CONCLUSION: This case suggested that immunosuppressive treatment enhanced with rituximab promoted the emergence of an HBV mutant within the determinant region of the S antigen, which escaped HBIg immunoprophylaxis causing HBV reactivation in a kidney transplant recipient.

Microbiological and molecular characteristics of carbapenemase-producing Klebsiella pneumoniae endemic in a tertiary Greek hospital during 2004-2010.

We report 570 carbapenemase-producing Klebsiella pneumoniae (CPKP) clinical isolates in a 1,040-bed Greek tertiary hospital during 2004 to 2010. The first CPKP (VIM-producing) was isolated in September 2004. Despite initial containment, VIM producers have become endemic since 2006. KPC-producing K. pneumoniae was first isolated in August 2007 from a patient who came from Israel, spread rapidly, and outcompeted VIM. Overall, 267 (47%) VIM-producing and 301 (53%) KPC-producing strains were isolated, including 141 (24.7%) from patients with bacteraemia. Two isolates carrying both VIM and KPC were isolated in two consecutive months in 2009, but not since. The prevalence of CPKP increased from 0% in 2003 to 38.3% in 2010 (p<0.0001). All genotyped KPC producers harboured blaKPC-2 and belonged to two clones, among which the hyperepidemic Greek clone, related to those from the United States and Israel, predominated. Most metallo-beta-lactamase (MBL) producers carried the blaVIM-1 gene and belonged to several clones, whereas all but one isolate with blaVIM-12 were clustered within a five-month period, arising from one clone. Resistance to non-beta-lactam antibiotics was also increased among CPKP. They were almost invariably resistant to ciprofloxacin and trimethoprim-sulfamethoxazole. Resistance to colistin increased from 3.5% (4/115) in 2008 to 20.8% (25/120) in 2010, and resistance to tigecycline also increased. Following reinforcement of infection control measures, prevalence of CPKP (mainly KPC) has been reduced since mid-2009 (from 46% in 2009 to 38.3% in 2010). In view of the exhaustion of available therapies, investment in infection control resources and optimal antibiotic use is urgently required.

Trimebutine as a potential antimicrobial agent: a preliminary in vitro approach.

AIM: The aim of this preliminary study was to investigate the in vitro effect of "non-antibiotic" trimebutine against reference strains Staphylococcus aureus ATCC 29213, ATCC 25923, Escherichia coli ATCC 25922, ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212; microbiota that are potentially involved in the pathophysiology of post-infectious functional gastrointestinal disorders. METHODS: Trimebutine activity was assessed by the broth microdilution method according to Clinical and Laboratory Standards Institute recommendations against reference strains S. aureus ATCC 29213 and ATCC 25923, E. coli ATCC 25922 and ATCC 35218, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212. Bactericidal activity of the compound was determined by spreading a 10 µL aliquot on Mueller-Hinton agar from each dilution showing non-visible growth. All tests were carried out in triplicate. RESULTS: Trimebutine was active against all strains tested presenting with MIC ranging from 1024 to 4000 mg/L. MIC and MBC were similar for E. coli ATCC 25922 and P. aeruginosa ATCC 27853 whereas for Gram-positive isolates and E. coli ATCC 35218 the MBC was higher. CONCLUSIONS: We demonstrated the in vitro bacteriostatic/bactericidal activity of trimebutine against bacteria frequently colonizing the gastrointestinal tract and potentially involved in human gastrointestinal infections that might trigger post-infectious functional gastrointestinal disorders.