Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as A. calcoaceticus, A. pittii and A. nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A.calcoaceticus -A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI -TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI -TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI -TOF mass spectra.
A monitoring effort that spanned across 1.5 years was conducted to examine three types of produce‐associated microbiota. The average amount of antibiotic‐resistant bacteria recovered from lettuce, tomato, and cucumber was 1.02 × 1010, 2.05 × 107, and 4.78 × 109 cells per 50 g of each produce, respectively. A total of 480 bacterial isolates were obtained and identified from their 16S rRNA genes, revealing isolates that were ubiquitously recovered from all three types of produce. However, sporadic presence of Klebsiella pneumoniae and Acinetobacter baumannii was detected on lettuce and cucumbers but not tomatoes. End‐point PCR revealed that the K. pneumoniae and A. baumannii isolates were positive for genes encoding extended spectrum beta‐lactamase. Whole genome sequencing of two of the K. pneumoniae isolates further suggested the presence of the blaCTX‐M‐15 gene in a conjugative plasmid, as well as other antibiotic resistance genes and virulence‐associated traits in either conjugative plasmids or the chromosomal genome. Quantitative microbial risk assessment indicated varying levels of ingestion risk associated with different types of produce. In particular, the risk arising from ESBL‐positive K. pneumoniae in lettuce, but not in cucumbers or tomatoes, was higher than the acceptable annual risk of 10−4.
Practical applications
Three types of vegetables were sampled and evaluated over 1.5 years to determine differences in their associated bacterial isolates. Particular emphasis was placed on identifying pathogenic strains that were positive for extended spectrum beta‐lactamase (ESBL). Quantitative estimates of the microbial risk associated with the ESBL‐positive pathogens showed that different produce types may incur varying levels of ingestion risk. Most of the currently reported ESBL‐positive bacterial isolates have been identified in nosocomial environments. However, the carriage of such drug‐resistant bacteria in vegetables suggests a possible connection between our daily diet and human health.
It has been described that the sensitivity of the Carba NP test may be low in the case of OXA-48-like carbapenamases and mass spectrometry based methods as well as a colorimetry based method have been described as alternatives. We evaluated 84 Enterobacteriaceae isolates including 31 OXA-48-like producing isolates and 13 isolates that produced either an imipenemase (IMP; n=8), New Delhi metallo-β-lactamase (NDM; n=3), or Klebsiella pneumoniae carbapenemase (KPC; n=2), as well as 40 carbapenemase negative Enterobacteriaceae isolates. We used the Neo-Rapid CARB kit, assessing the results with the unaided eye and compared it with a colorimetric approach. Furthermore, we incubated the isolates in growth media with meropenem and measured the remaining meropenem after one and 2h of incubation, respectively, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Whilst all carbapenemase producing isolates with the exception of the OXA-244 producer tested positive for both the Neo-rapid CARB test using the unaided eye or colorimetry, and the 13 isolates producing either IMP, NDM or KPC hydrolysed the meropenem in the media almost completely after 2h of incubation, the 31 OXA-48-like producing isolates exhibited very variable hydrolytic activity when incubated in growth media with meropenem. In our study, the Neo-Rapid CARB test yielded a sensitivity of 98% for both the traditional and the colorimetric approach with a specificity of 95% and 100% respectively. Our results indicate that the Neo-Rapid CARB test may have use for the detection of OXA-48 type carbapenemases and that it may be particularly important to ensure bacterial lysis for the detection of these weaker hydrolysers.
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