Objectives: To assess the prevalence and types of genes encoding extended-spectrum β-lactamases (ESBLs) in clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. from Bolivia. Methods: A total of 642 clinical isolates were collected consecutively during a 4 month period (September to December 2004). Resistance or reduced susceptibility to cefotaxime and/or ceftazidime and/or aztreonam was assessed using double disc synergy tests using clavulanic acid, cefotaxime, ceftazidime and aztreonam to identify putative ESBL-producing isolates. The ESBL determinants were characterized by colony blot hybridization, PCR and DNA sequencing. Results: Of the 642 isolates, 220 (34.3%) showed resistance or reduced susceptibility to cefotaxime and/or ceftazidime and/or aztreonam, and 150 (23.4%) were putative ESBL producers. A total of 106 ESBL-producing isolates contained the bla_CTX-M-2 gene, and 32 isolates had a novel allele, bla_CTX-M-43. bla_CTX-M alleles were detected in all P. aeruginosa and Acinetobacter spp. studied. In contrast, only 12 ESBL-producing isolates had bla_PER-2, mainly Enterobacteriaceae, although it was also found in a strain of P. aeruginosa. Conclusions: This is the first study on ESBL-producing strains in Bolivia and it reveals a high prevalence of bla_CTX-M genes. The PER-2 enzyme was less prevalent, but its gene was detected in several species, including P. aeruginosa, which is consistent with horizontal transfer.

Infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) have limited therapeutic options. Sulbactam-durlobactam is a combination of two βlactamase inhibitors with activity against CRAB under phase 3 clinical investigation. We performed a systematic review on in vitro studies reporting A. baumannii resistances against sulbactam/durlobactam. We considered “resistant” species to be those with MIC ≥ 8 mg/L. Ten studies were included in the review (9754 tested isolates). Overall, 2.3% of A. baumannii were resistant to sulbactam/durlobactam, and this percentage rose to 3.4% among CRAB subgroups and to 3.7% among colistin-resistant strains. Resistance was 100% among metallo β-lactamase-producing strains. Overall, in 12.5% of cases, sulbactam/durlobactam resistance was associated with the production of NDM-1, in 31.7% of cases with the substitutions in the PBP3 determinants, and in the remaining cases the resistance mechanism was unknown. In conclusion, A. baumannii resistance towards sulbactam/durlobactam is limited, except for MBL-producing strains.

The ability to form biofilms is a recognized trait of Stenotrophomonas maltophilia, but the extent of its clinical relevance is still unclear. The present multicenter prospective study (ANSELM) aims at investigating the association between biofilm formation and clinical outcomes of S. maltophilia infections. One hundred and nine isolates were collected from various geographical origins and stratified according to their clinical relevance. Biofilm formation was evaluated by the microtiter plate assay and correlated with microbiological and clinical data from the associated strains. Antibiotic susceptibility of the planktonic cells was tested by the disk diffusion technique, while antibiotic activity against mature biofilms was spectrophotometrically assessed. Most strains (91.7%) were able to form biofilm, although bloodborne strains produced biofilm amounts significantly higher than strains causing hospital- rather than community-acquired infections, and those recognized as “definite” pathogens. Biofilm formation efficiency was positively correlated with mechanical ventilation (p = 0.032), whereas a negative relationship was found with antibiotic resistance (r² = 0.107; p < 0.001), specifically in the case of the pathogenic strains. Mature S. maltophilia biofilms were markedly more resistant (up to 128 times) to cotrimoxazole and levofloxacin compared with their planktonic counterparts, especially in the case of bloodborne strains. Our findings indicate that biofilm formation by S. maltophilia is obviously a contributing factor in the pathogenesis of infections, especially in deep ones, thus warranting additional studies with larger cohort of patients and isolates.

The aim of the study was the biochemical characterization of a new variant of the metallo-β-lactamase, IMP-22. Moreover, the genetic environment of the bla_IMP-22 gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L’Aquila, Italy. Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (K_m and V_max) were determined for a large pattern of substrates. A new IMP metallo-β-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The bla_IMP-22 encodes for a pre-protein of 246 amino acids and the N-terminus of the mature β-lactamase (NH₂-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (K_m and V_max), IMP-22 was found to hydrolyse narrow- and extended-spectrum β-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC₅₀ of 800, 750 and 300 µM, respectively. The finding of the bla_IMP-22 gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of bla_MBL genes in several bacterial species and in different environments.

This study investigated the genetic environment of bla CTX-M genes and associated resistance genes in seven Proteus mirabilis and six Morganella morganii extended spectrum β-lactamase (ESBL)-positive isolates. The isolates were recovered from hospitalized patients with respiratory or urinary tract infections at the Military Hospital of Tunis, Tunisia. Twenty-one of the 200 strains exhibited non-susceptibility to third generation cephalosporins and, among these strains, the double-disk synergy test confirmed the ESBL phenotype in 13 isolates. These ESBL producers were co-resistant to chloramphenicol, tetracycline and oflaxacin but remained susceptible to ertapenem (MIC<0.25 mg l−1). The presence and nature of bla CTX-M-15, bla CTX-M-8, bla TEM-24, bla TEM-1 and bla TEM-2 genes was determined by PCR and sequencing. Chromosomal localization of the bla CTX-M-15 gene was confirmed in all strains, with the exception of M. morganii isolate M-17991, by Southern-blot analysis performed either on chromosomal or plasmid DNA. M. morganii M-12012 and M. morganii M-6019 showed the same PFGE pattern, whereas the remaining CTX-M-15-producing isolates were unrelated. The presence of ISEcp1 was ascertained in CTX-M-15-producing isolates. A class 1 integron with different gene cassettes (dfrA1, orfC and aadB) was found in five P. mirabilis and six M. morganii isolates.