Cecilia G. Carvalhaes

Multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii: resistance mechanisms and implications for therapy

Pseudomonas aeruginosa and Acinetobacter baumannii are major nosocomial pathogens worldwide. Both are intrinsically resistant to many drugs and are able to become resistant to virtually any antimicrobial agent. An increasing prevalence of infections caused by multidrug-resistant (MDR) isolates has been reported in many countries. The resistance mechanisms of P. aeruginosa and A. baumannii include the production of β-lactamases, efflux pumps, and target-site or outer membrane modifications. Resistance to multiple drugs is usually the result of the combination of different mechanisms in a single isolate or the action of a single potent resistance mechanism. There are many challenges in the treatment of MDR P. aeruginosa and A. baumannii, especially considering the absence of new antimicrobials in the drug-development pipeline. In this review, we present the major resistance mechanisms of P. aeruginosa and A. baumannii, and discuss how they can affect antimicrobial therapy, considering recent clinical, microbiological, pharmacokinetic and pharmacodynamic findings of the main drugs used to treat MDR isolates.

Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results

OBJECTIVES The aim of this study was to evaluate the presence of carbapenemases in a Klebsiella pneumoniae collection and the performance of the modified Hodge test (MHT) to correctly identify this phenotype. METHODS Twenty-eight K. pneumoniae clinical isolates with reduced susceptibility to carbapenems were evaluated. Antimicrobial susceptibility and molecular typing were performed by agar dilution and PFGE, respectively. The MHT was performed using both standard and high inoculum of test organisms. Imipenem hydrolysis was investigated by spectrophotometric assays and carbapenemase-encoding genes were identified by PCR and amplicon sequencing. Porin loss was investigated by both PCR and SDS-PAGE. RESULTS Susceptibility rates for imipenem, meropenem and ertapenem were 93%, 57% and 11%, respectively. The PFGE analysis showed seven unrelated genotypes. By testing standard inoculum and ertapenem or meropenem discs, 25% (n = 7) and 21% (n = 6) of the isolates were classified as carbapenemase producers, respectively. When a higher inoculum was employed, these rates increased to 54% (n = 15) and 43% (n = 12), respectively. No imipenem hydrolysis was detected. PCRs identified bla(CTX-M) in 27 (96%) isolates, of which 2 isolates also carried bla(GES-1.) SDS-PAGE and PCR assays revealed that all isolates had lost at least one outer membrane protein, except for a single isolate that was found to express both OmpK35 and OmpK36. CONCLUSIONS False detection of carbapenemase production was observed by the MHT possibly as a result of extended-spectrum beta-lactamase (ESBL) production coupled with porin loss as reported before. Clinical laboratories must be aware of this fact, especially in geographical areas where ESBL-producing isolates are highly prevalent.

OXA-72-producing Acinetobacter baumannii in Brazil: a case report

by the integration of a partly truncated class 2 integron as well as the deletion of large parts of the mobA gene (including the mobB gene) and the tetracycline resistance gene tet(B). To the best of our knowledge, this is the first description of class 2-associated resistance gene cassettes in members of the genus Pasteurella. In contrast to P. multocida, which is found in the respiratory tract of various animal species, P. aerogenes is commonly found in the intestinal tract of swine, and may be associated with diarrhoea and other infections. 5 As such, it is in contact with Enterobacteriaceae, which frequently carry class 1 and class 2 integrons on plasmids. 9 Since plasmids from E. coli and other Enterobacteriaceae usually cannot replicate in Pasteu-rella hosts, recombination of a transferred enterobacterial plasmid with plasmids indigenous to Pasteurella ensures a stable maintenance of the newly acquired resistance genes in the Pasteurella host. The in-depth analysis of plasmid pCCK343 from P. aerogenes provides further support for such inter-and intra-plasmid recombination events. Moreover, the data presented in this study substantiate the assumption of a gene flux between Enterobacteriaceae and P. aerogenes, which has previously been suggested based on the finding that the tetracycline resistance gene tet(B)—the predominant tet gene among Enterobacteriaceae—is most frequently found in P. aerogenes.

Detection of SPM-1-Producing Pseudomonas aeruginosa and Class D β-Lactamase-Producing Acinetobacter baumannii Isolates by Use of Liquid Chromatography-Mass Spectrometry and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT This study evaluates the accuracy of liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for detecting carbapenem hydrolytic activity among SPM-1-, GIM-1-, and GES-5-producing Pseudomonas aeruginosa isolates and OXA-143-, IMP-10-, and OXA-58-producing Acinetobacter baumannii isolates. Class A and B carbapenemase activities were rapidly detected by MALDI-TOF in a 2-h assay. However, an extended incubation time was necessary for detection of carbapenem-hydrolyzing class D β-lactamase (CHDL) activity in Acinetobacter spp.

Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision

OBJECTIVES Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was successfully applied for the detection of carbapenemase activity directly from Gram-negative colonies. Based on this principle, we evaluated the performance of MALDI-TOF MS for rapid detection of carbapenemase activity directly from positive blood culture vials. METHODS A total of 100 blood culture vials were randomly selected. MALDI-TOF MS carbapenemase assay results were confirmed by the detection of carbapenemase-encoding genes. RESULTS A total of 110 bacterial isolates were recovered. The MALDI-TOF MS carbapenemase assay identified 21 of 29 (72.4%) of the carbapenemase-producing isolates directly from the blood culture vials, especially those encoding KPC-2 (100%) and SPM-1 (100%), after a 4 h incubation period. Although the majority of OXA-23-producing Acinetobacter baumannii isolates were not identified on day 1, all isolates were identified as carbapenemase producers directly from the colony on the next day. CONCLUSIONS The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.

Outbreak of Carbapenem-Resistant Providencia stuartii in an Intensive Care Unit

Outbreaks by carbapenem-resistant Providencia stuartii (CRPS) are rarely described. Clinical characteristics of patients with CRPS in an intensive care unit and resistance mechanisms were investigated. Carbapenemase production and/or outer membrane alterations were not detected; only CTX-M-2 and AmpC hyperproduction were noted. The outbreak was ultimately controlled in a 3-month period.

Coproduction of KPC-2 and IMP-10 in Carbapenem-Resistant Serratia marcescens Isolates from an Outbreak in a Brazilian Teaching Hospital

ABSTRACT We describe an outbreak caused by KPC-2- and IMP-10-producing Serratia marcescens isolates in a Brazilian teaching hospital. Tigecycline was the only active antimicrobial agent tested. The bla IMP-10 gene was located in a new class 1 integron, named In990, carried by a nonconjugative plasmid, in contrast to bla KPC-2.

Identification of a New Integron Harboring blaIMP-10 in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

We describe herein the characterization of a novel class 1 integron harboring the bla IMP-10 gene in Acinetobacter baumannii clinical isolates.…

Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae in the intensive care unit: a real challenge to physicians, scientific community, and society.

ABSTRACT The prevalence of multidrug-resistant pathogens, especially in intensive care units, has increased and represents a great concern for medical and scientific community. Infections caused by these pathogens are associated with increased costs, length of hospitalization, and morbidity/mortality rates. The last decade was marked by the spread of carbapenem resistance determinants especially in Enterobacteriaceae isolates. In this review, the Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae is used as an example to discuss the difficulties in dealing with multidrug-resistant pathogens in the intensive care unit setting and how they represent a challenge to the medical-scientific community and, ultimately, the whole society.

Influence of Culture Media on Detection of Carbapenem Hydrolysis by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT In this study, we evaluated the influence of distinct bacterial growth media on detection of carbapenemase hydrolysis by matrix-assisted laser desorption ionization–time of flight mass spectrometry. False-negative results were observed for OXA-25-, OXA-26-, and OXA-72-producing Acinetobacter baumannii isolates grown on MacConkey agar medium. The other culture media showed 100% sensitivity and 100% specificity for detecting carbapenemase.