Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa.

Carbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.

Sub-MIC effects of a proline-rich antibacterial peptide on clinical isolates of Acinetobacter baumannii.

INTRODUCTION: Acinetobacter baumannii is one of the most important nosocomial pathogens, mainly due to its ability to accumulate antibiotic-resistances and to persist in the hospital environment - characteristics related to biofilm production. It is well-known that A. baumannii is inhibited by the proline-rich peptide Bac7(1-35), but its putative effects at sub-MICs were never considered. AIMS: We examined the sub-MIC effect of Bac7(1-35) on the growth rate, resistance induction and some A. baumannii features linked to virulence. METHODOLOGY: Growth kinetics in the presence of sub-MICs of Bac7(1-35) were evaluated spectrophotometrically. Peptide uptake was quantified by cytometric analysis. The ability of Bac7(1-35) to interfere with biofilm production was investigated by the crystal violet method and confocal microscopy. Bacterial motility was observed at the interphase between a layer of a semi-solid medium and the polystyrene bottom of a Petri dish. The induction of resistance was evaluated after serial passages with sub-MICs of the peptide. RESULTS: Although the MIC of Bac7(1-35) was between 2-4 µM for all tested strains, its effect on the growth rate at sub-MICs was strain-dependent and correlated with the amount of peptide internalized by each strain. Sub-MICs of Bac7(1-35) induced a strongly strain-dependent effect on biofilm formation and reduced motility in almost all strains, but interestingly the peptide did not induce resistance. CONCLUSION: Bac7(1-35) is internalized into A. baumannii and is able to inhibit biofilm formation and bacterial motility, without inducing resistance. This study stresses the importance of considering possible effects that antimicrobials could have at sub-MICs, mimicking a common condition during antibiotic treatment.

Biofilms Developed on Dental Implant Titanium Surfaces with Different Roughness: Comparison Between In Vitro and In Vivo Studies.

Microbial biofilms developed on dental implants play a major role in perimplantitis' pathogenesis. Many studies have indicated that surface roughness is the main feature favoring biofilm development in vitro, but its actual influence in vivo has still to be confirmed. In this study, the amount of biofilm formed on differently treated titanium surfaces, showing distinct roughness, has been examined both in vivo and in vitro by Confocal Laser Scanning Microscopy. In vitro studies availed of biofilm developed by Pseudomonas aeruginosa or by salivary bacteria from volunteer donors. In vivo biofilm production was obtained by exposing titanium discs to the oral cavity of healthy volunteers. In vitro experiments showed that P. aeruginosa and, to a lesser extent, salivary bacteria produce more biomass and develop thicker biofilms on laser-treated and sandblasted titanium surfaces with respect to machined ones. In vivo experiments confirmed that bacterial colonization starts on sites of surface unevenness, but failed to disclose biomass differences among biofilms formed on surfaces with different roughness. Our study revealed that biofilm developed in vitro is more easily influenced by surface features than biofilm formed by complex communities in the mouth, where the cooperation of a variety of bacterial species and the presence of a wide range of nutrients and conditions allow bacteria to optimize substrate colonization. Therefore, quantitative differences observed in vitro among surfaces with different characteristics may not be predictive of different colonization rates in vivo.

Spread of Vancomycin-Resistant Enterococcus faecium Isolates Despite Validated Infection Control Measures in an Italian Hospital: Antibiotic Resistance and Genotypic Characterization of the Endemic Strain.

An alarming increase of vancomycin-resistant Enterococcus faecium (VREfm) isolates was detected in an Italian referral hospital subjected to policies of infection control validated by the Joint Commission International. Analysis of the population structure of 122 consecutive, nonreplicate VREfm isolates collected over an 18-month period identified a single major clone that spread around the whole hospital, rapidly establishing an endemic state. It belonged to sequence type (ST) 17 and showed a highly multidrug-resistant phenotype, being resistant to all antimicrobial classes for the carriage of several resistance determinants. Furthermore, some strains with decreased susceptibility to daptomycin were detected. Eighteen out of the 122 isolates did not group in the major clone. They showed a low spreading potential inside the hospital wards, even if most of them displayed a multidrug-resistant phenotype and belonged to a hospital-adapted lineage. Causes that led to the VREfm endemic state have not been fully elucidated. However, it is conceivable that the increase in systemic antibiotic consumption and the use of selective digestive tract decontamination, including vancomycin in critically ill patients during the period before 2014, may have played a role in the ST17 clone dissemination, but additional traits conferring high fitness in hospital environment cannot be excluded.

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1-35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

Epidemic Dissemination of a Carbapenem-Resistant Acinetobacter baumannii Clone Carrying armA Two Years After Its First Isolation in an Italian Hospital.

This study describes the dissemination of a carbapenem-resistant Acinetobacter baumannii (CRAB) strain in a university hospital in Northeast Italy. Characterization of the outbreak strain was combined with a retrospective analysis of all CRAB isolates collected in the same hospital during the 5 years preceding the outbreak, with the aim of elucidating the origin of the epidemic spread. The outbreak strain was shown to belong to the International Clone II and carry the blaOXA-23 gene, flanked by two ISAba1 sequences in opposite orientation (Tn2006 arrangement). The epidemic clone harbored also the blaOXA-66 allele of the carbapenemase intrinsic to A. baumannii, the determinant of ArmA 16S rRNA methylase and a class 1 integron, with the aacA4, catB8, and aadA1 cassette array. Genotype analysis, performed by macrorestriction analysis and VRBA, revealed that isolates related to outbreak strain had been sporadically collected from inpatients in the 2 years preceding outbreak start. Carriage of blaOXA-66, armA, and the integron further supported relatedness of these isolates to the outbreak clone. Outbreak initially involved three medical wards, typically hosting elderly patients with a history of prolonged hospitalization. The study highlights the need to adopt strict infection control measures also when CRAB isolation appears to be a sporadic event.

pKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus.

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Metallo-ß-lactamase expression confers an advantage to Pseudomonas aeruginosa isolates compared with other ß-lactam resistance mechanisms, favoring the prevalence of metallo-ß-lactamase producers in a clinical environment.

The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings.

Colonization of the tip of a thoracic catheter by Enterococcus faecalis resistant to vancomycin and linezolid.

We report the isolation of Enterococcus faecalis resistant to vancomycin and linezolid from the tip of a thoracic drainage catheter in an elderly patient. He was treated with vancomycin for a pleural empyema due to a meticillin-resistant Staphylococcus aureus but never received linezolid. A surveillance rectal swab yielded both linezolid-susceptible and -resistant strains, and the two isolates were not genotypically related. Careful monitoring for linezolid-resistance is critical to avoid potential therapy failure and transmission of resistant E. faecalis.

Molecular evolution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a nosocomial setting of high-level endemicity.

An outbreak of multidrug-resistant Pseudomonas aeruginosa strains producing VIM-type metallo-beta-lactamases (MBLs) has occurred in an Italian hospital since 2000 (C. Lagatolla, E. A. Tonin, C. Monti-Bragadin, L. Dolzani, F. Gombac, C. Bearzi, E. Edalucci, F. Gionechetti, and G. M. Rossolini, Emerg. Infect. Dis. 10:535-538, 2004). In this work, using molecular methods, we characterized 128 carbapenem-resistant isolates (including 98 VIM-positive isolates) collected from that hospital from 2000 to 2002 to investigate the dynamics of the dissemination of MBL producers in the clinical setting. Genotyping by random amplification of polymorphic DNA and pulsed-field gel electrophoresis showed that most VIM-positive isolates belonged to two different clonal lineages, producing either a VIM-1- or a VIM-2-like MBL, whose ancestors were detected for the first time in the hospital in 1999, suggesting that clonal expansion played a predominant role in the dissemination of these isolates. The 86 clonally related isolates carrying a blaVIM-1-like gene on an In70-like integron were clearly related to a VIM-1-positive P. aeruginosa clone circulating in various Italian hospitals since the late 1990s. VIM-negative P. aeruginosa strains related to the VIM-1-positive clone were detected during the same period, suggesting that the latter strain was derived from a clonal lineage already circulating in the hospital. In the VIM-2-like positive clone, the MBL gene was carried by an unusual class 1 integron, named In71, lacking the 3' conserved sequence region typical of sul1-associated integrons. A different class 1 integron with an original structure carrying a blaVIM-2 determinant, named In74, was detected in a sporadic isolate. A retrospective investigation did not reveal the presence of strains related to any of the VIM-producing isolates earlier than 1997.

Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

Diversity of aminoglycoside-resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones.

The purpose of the present study was to investigate the diversity of the genes encoding aminoglycoside-modifying enzymes and their association with class 1 integrons in three pan-European clones of Acinetobacter baumannii. The study collection included 106 multidrug-resistant strains previously allocated to clone I (n = 56), clone II (n = 36) and clone III (n = 6) and a heterogeneous group of other strains (n = 8), using AFLP fingerprinting and ribotyping. The strains were from hospitals of the Czech Republic (n = 70; collected 1991-2001) and 12 other European countries (n = 36; 1982-1998). Using PCR, at least one of the following aminoglycoside-resistance genes was detected in 101 (95 %) strains: aphA1 (n = 76), aacC1 (n = 68), aadA1 (n = 68), aphA6 (n = 55), aadB (n = 31), aacC2 (n = 7) and aacA4 (n = 3). A combination of two to five different resistance genes was observed in 89 strains (84 %), with a total of 12 different combinations. PCR mapping revealed that aacC1, aadA1 and aacA4 were each associated with a class 1 integron, as was the case with aadB for six strains of clone III. Six different class 1 integron variable regions were detected in 78 strains (74 %), with two predominant regions (2.5 and 3.0 kb) in two sets of 34 strains each. The 3.0 kb region contained five gene cassettes (aacC1, orfX, orfX, orfX', aadA1) and differed from the 2.5 bp region only by one additional orfX cassette. These two integron regions were confined to clones I and II and were found in strains isolated in seven countries between 1982 and 2001. The clone III strains were homogeneous both in resistance genes and in integron variable regions, whereas clones I and II showed a remarkable intraclonal diversity of these properties, with no clear-cut difference between the two clones. Yet, within the Czech clone I and II strains, the diversity of resistance genes and integron structures was limited as compared to those from other countries. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.

Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates.

Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity. At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria. The fragments Bac7(1-35) and, to a lesser extent, Bac7(1-16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia. In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.

Endemic carbapenem-resistant Pseudomonas aeruginosa with acquired metallo-beta-lactamase determinants in European hospital.

Acquired metallo-beta-lactamases (MBLs) can confer broad-spectrum beta-lactam resistance (including carbapenems) not reversible by conventional beta-lactamase inhibitors and are emerging resistance determinants of remarkable clinical importance. In 2001, multidrug-resistant Pseudomonas aeruginosa carrying bla(VIM) MBL genes were found to be widespread (approximately 20% of all P. aeruginosa isolates and 70% of the carbapenem-resistant isolates) at Trieste University Hospital. Clonal diversity and heterogeneity of resistance determinants (either bla(VIM-1)-like or bla(VIM-2)-like) were detected among MBL producers. This evidence is the first that acquired MBLs can rapidly emerge and establish a condition of endemicity in certain epidemiologic settings.

Molecular characterization of integrons in epidemiologically unrelated clinical isolates of Acinetobacter baumannii from Italian hospitals reveals a limited diversity of gene cassette arrays.

Integron carriage by 36 epidemiologically unrelated Acinetobacter baumannii isolates collected over an 11-year period from patients in six different Italian hospitals was investigated. Sixteen type 1 integron-positive isolates (44%) were found, 13 of which carried the same array of cassettes, i.e., aacC1, orfX, orfX', and aadA1a. As ribotype analysis of the isolates demonstrated a notable genetic diversity, horizontal transfer of the entire integron structure or ancient acquisition was hypothesized.

Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced.

Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab. They were biochemically identified using API20NE and confirmed by a PCR-based assay. The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV. All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis. Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure. The polysaccharides were analysed by means of methylation analysis and 1H-NMR spectroscopy in order to determine structural similarities. It was shown that different strains are capable of producing chemically different polysaccharides.