Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. A major factor contributing to success of this bacterium is its outstanding ability to survive on dry surfaces. The molecular basis for desiccation resistance is not completely understood. This study focused on growth under osmotic stress and aimed to identify the pool of compatible solutes synthesized in response to these low water activity conditions. A. baumannii produced mannitol as compatible solute, but in contrast to Acinetobacter baylyi, also trehalose was accumulated in response to increasing NaCl concentrations. The genome of A. baumannii encodes a trehalose-6-phosphate phosphatase (OtsB) and a trehalose-6-phosphate synthase (OtsA). Deletion of otsB abolished trehalose formation, demonstrating that otsB is essential for trehalose biosynthesis. Growth of the mutant was neither impaired at low salt nor at 500 mM NaCl, but it did not grow at high temperatures, indicating a dual function of trehalose in osmo- and thermoprotection. This led us to analyse temperature dependence of trehalose formation. Indeed, expression of otsB was not only induced by high osmolarity but also by high temperature. Concurrently, trehalose was accumulated in cells grown at high temperature. Taken together, these data point to an important role of trehalose in A. baumannii beyond osmoprotection.

Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid (∼40 min) real-time PCR assays targeting the most common extended spectrum β-lactamases (ESBLs; blaCTX-M-1 and blaCTX-M-9 groups, plus blaTEM, blaSHV, and an internal 16S ribosomal DNA control). AmpC CMY β-lactamase (blaCMY) prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one β-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and 17961.

27Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major 28 threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates 29 from 258 patients presenting at a regional Australian hospital over a 14-month period. 30Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates 31 were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most 32 common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, 33 SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase 34 prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R 35 isolates obtained from the intensive care unit, were subjected to whole-genome sequencing 36 (WGS) to assess transmission dynamics, the presence of unidentified resistance 37 determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae 38 (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter 39 cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% 40 and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings 41 in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase 42 gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 43 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY genes. WGS of β-lactamase negative or 44 carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including45 blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission 46 among intensive care unit patients was identified. We demonstrate that our PCR assays 47 enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has 48 important infection control and therapeutic implications.49 52 determinants within and among bacterial populations [1]. Both infection and colonization 53 with these globally-disseminated organisms are associated with poor clinical outcomes and 54 death [2]. 3GC-R Enterobacteriaceae prevalence rates vary considerably among hospitals 55 and countries, with particularly high rates in India, Asia, and the Middle East [3]. Australia 56 has historically observed a relatively low frequency of 3GC-R Enterobacteriaceae. However, 57 this pattern may be changing, with an extended spectrum β-lactamase (ESBL) phenotype 58 detected in 13.3% of E. coli and 9.8% of K. pneumoniae in blood culture isolates in 2018 [4], 59 up from 2013 rates of 7.6% and 6.3%, respectively. Moreover, ceftriaxone resistance was 60 seen in 13.4% of E. coli and 9.4% of K. pneumoniae, with 86.3% and 82.6% containing CTX-M-61 type ESBL genes, respectively [4]. 62 63 AMR in 3GC-R Enterobacteriaceae is generally conferred by the presence of an ESBL or the 64 over-expression of a chromosomal or plasmid-borne AmpC β-lactamase; however, the 65 prevalence of these determinants var...