Phenotypic detection of clinical isolates of Haemophilus influenzae with altered penicillin-binding protein 3.

The aims of this study were to determine the correlation of mutations in the ftsI gene (coding for PBP3) of Haemophilus influenzae with aminopenicillin resistance and to evaluate the 2017 European Committee for Antibiotic Susceptibility Testing (EUCAST) guidelines for clinical categorization of ampicillin, amoxicillin, and amoxicillin-clavulanate for strains with mutated PBP3 conferring resistance (rPBP3). A panel of 91 H. influenzae isolates was genetically characterized by sequencing of the fstI gene. For all the studied isolates, a screening with benzylpenicillin 1U (BP1) was carried out and minimum inhibitory concentrations (MICs) of ampicillin, amoxicillin, and amoxicillin-clavulanate were tested and interpreted according to EUCAST recommendations. ftsI sequence analysis revealed a total of 14 different amino acid substitutions in PBP3. The substitution patterns most commonly observed were [D350N, M377I, A502V, N526K] among the bla-positive rPBP3 strains (37.5%) and [D350N, A502T, N526K] among the bla-negative rPBP3 strains (24.5%). Screening with BP1 was able to correctly categorize 100% of the bla-negative sPBP3 strains, 100% of the bla-positive strains, and 92% of the bla-negative rPBP3 ones. Only 29% of the bla-negative rPBP3 strains evaluated displayed ampicillin MICs above the current EUCAST resistant breakpoint defined at 1 µg/ml. The PBP3 substitution patterns of the strains evaluated are similar to the ones observed in previous Spanish and European studies. Although the screening with BP1 proved to be adequate in the detection of bla-negative rPBP3 strains, these cannot be reliably identified by current 2018 EUCAST breakpoints for ampicillin.

Evaluation of the carbapenem inactivation method (CIM) for detecting carbapenemase activity in enterobacteria.

OBJECTIVES: The objective of this study was to evaluate the accuracy of the CIM test in the detection of carbapenemase activity in 124 strains of Enterobacteriaceae. METHODS: A panel of 124 previously characterized Enterobacteriaceae was tested: 77 strains producing the following carbapenemase families: KPC (n = 14), GES (n = 22), NDM (n = 19), VIM (n = 4), IMP (n = 4) and OXA-48 (n = 14) and 47 non-carbapenemase producers. For the CIM method, an active susceptibility meropenem disc was exposed to a bacterial suspension of a test strain; when a carbapenemase is produced, the antibiotic is inactivated allowing uninhibited growth of an indicator strain after overnight incubation. A clear inhibition zone (≥20 mm) was considered indicative of no-carbapenemase activity. RESULTS: All KPC, NDM, VIM, IMP or OXA-48 producing strains were unequivocally detected with the CIM test. CIM false negative results were obtained with eleven Enterobacter cloacae producing GES-6. Two other E. cloacae not producing carbapenemase (one with SHV-12, one hyperproducing AmpC) were positive by the test. The sensitivity and specificity of the assay compared to those of molecular methods were 85.7% and 95.7%, respectively. CONCLUSIONS: The CIM method proved to be inexpensive and easy to interpret. It provided less than optimal results in the detection of GES-6 activity.

Non-molecular detection of carbapenemases in Enterobacteriaceae clinical isolates.

Infections caused by carbapenemase-producing bacteria are becoming a major clinical and public health concern. Detection of carbapenemase producing strains is often challenging, since susceptibility to carbapenems may vary significantly among carbapenemase producers. Some carbapenemases have shown to exhibit weak activity against carbapenems leading to minimum inhibitory concentrations of carbapenems below the breakpoint for defining clinical resistance and even below the proposed screening breakpoint. Thus, reliable and rapid detection of carbapenemase-activity is needed for an appropriate patient management and a rapid implementation of infection prevention and control measures. Over the last years, an increasing number of non-molecular assays for prompt detection of carbapenemase activity have been described and developed. However, none of the currently available phenotypic methods have proved to be full specific and sensitive. Selection of the appropriate methodological approach will depend on each situation. Factors to be considered include the epidemiological status, laboratory resources and availability of other confirmation tests. In this review, we provide an overview of the currently available non-molecular methods for detection of carbapenemase activity.

[Correlation between MALDI-TOF Vitek-MSTM system and conventional identification methods of gastrointestinal infection causing bacteria]. / Correlación entre el sistema de MALDI-TOF Vitek-MSTM y los métodos convencionales de identificación de bacterias causantes de infección gastrointestinal.

OBJECTIVE: Rapid identification of pathogens is essential for the diagnosis of gastrointestinal infections. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry has shown to be effective and fast for the identification of microorganisms. The objective of this study was to evaluate the correlation between Vitek-MSTM and conventional methods for bacterial identification causing gastrointestinal infection. METHODS: A total of 329 gastrointestinal pathogens were identified using Vitek-MSTM (v2 SARAMIS MS -ID, bioMérieux, Marcy-I´Étoile, France) and routine diagnostic methods simultaneously. In cases of discrepancy 16SrRNA gene sequencing was performed. RESULTS: The correlation between Vitek-MSTM and diagnostic methods was 100% except for Yersinia enterocolitica (94.1%), Helicobacter pylori (10%) and Aeromonas veronii (0 %). CONCLUSIONS: Vitek-MSTM is a quick and useful method for identification of enterophatogenic bacteria. It is necessary to improve the performance of the system for the identification of H. pylori and A. veronii.