Investigation of a Candida guilliermondii Pseudo-outbreak Reveals a Novel Source of Laboratory Contamination.

Candida guilliermondii was isolated from sterile specimens with increasing frequency over a several-month period despite a paucity of clinical evidence suggesting true Candida infections. However, a health care-associated outbreak was strongly considered due to growth patterns in the microbiology laboratory that were more consistent with true infection than environmental contamination. Therefore, an extensive investigation was performed to identify its cause. With the exception of one case, patient clinical courses were not consistent with true invasive fungal infections. Furthermore, no epidemiologic link between patients was identified. Rather, extensive environmental sampling revealed C. guilliermondii in an anaerobic holding jar in the clinical microbiology laboratory, where anaerobic plates were prereduced and held before inoculating specimens. C. guilliermondii grows poorly under anaerobic conditions. Thus, we postulate that anaerobic plates became intermittently contaminated. Passaging from intermittently contaminated anaerobic plates to primary quadrants of aerobic media during specimen planting yielded a colonial growth pattern typical for true specimen infection, thus obscuring laboratory contamination. A molecular evaluation of the C. guilliermondii isolates confirmed a common source for pseudo-outbreak cases but not for the one true infection. In line with Reason's model of organizational accidents, active and latent errors coincided to contribute to the pseudo-outbreak. These included organism factors (lack of growth in anaerobic conditions obscuring plate contamination), human factors (lack of strict adherence to plating order, leading to only intermittent observation of aerobic plate positivity), and laboratory factors (novel equipment). All of these variables should be considered when evaluating possible laboratory-based pseudo-outbreaks.

Whole-genome typing and characterization of blaVIM19-harbouring ST383 Klebsiella pneumoniae by PFGE, whole-genome mapping and WGS.

OBJECTIVES: We utilized whole-genome mapping (WGM) and WGS to characterize 12 clinical carbapenem-resistant Klebsiella pneumoniae strains (TGH1-TGH12). METHODS: All strains were screened for carbapenemase genes by PCR, and typed by MLST, PFGE (XbaI) and WGM (AflII) (OpGen, USA). WGS (Illumina) was performed on TGH8 and TGH10. Reads were de novo assembled and annotated [SPAdes, Rapid Annotation Subsystem Technology (RAST)]. Contigs were aligned directly, and after in silico AflII restriction, with corresponding WGMs (MapSolver, OpGen; BioNumerics, Applied Maths). RESULTS: All 12 strains were ST383. Of the 12 strains, 11 were carbapenem resistant, 7 harboured blaKPC-2 and 11 harboured blaVIM-19. Varying the parameters for assigning WGM clusters showed that these were comparable to STs and to the eight PFGE types or subtypes (difference of three or more bands). A 95% similarity coefficient assigned all 12 WGMs to a single cluster, whereas a 99% similarity coefficient (or ≥10 unmatched-fragment difference) assigned the 12 WGMs to eight (sub)clusters. Based on a difference of three or more bands between PFGE profiles, the Simpson's diversity indices (SDIs) of WGM (0.94, Jackknife pseudo-values CI: 0.883-0.996) and PFGE (0.93, Jackknife pseudo-values CI: 0.828-1.000) were similar (P = 0.649). However, the discriminatory power of WGM was significantly higher (SDI: 0.94, Jackknife pseudo-values CI: 0.883-0.996) than that of PFGE profiles typed on a difference of seven or more bands (SDI: 0.53, Jackknife pseudo-values CI: 0.212-0.849) (P = 0.007). CONCLUSIONS: This study demonstrates the application of WGM to understanding the epidemiology of hospital-associated K. pneumoniae. Utilizing a combination of WGM and WGS, we also present here the first longitudinal genomic characterization of the highly dynamic carbapenem-resistant ST383 K. pneumoniae clone that is rapidly gaining importance in Europe.

Comparative whole-genome mapping to determine Staphylococcus aureus genome size, virulence motifs, and clonality.

Despite being a clonal pathogen, Staphylococcus aureus continues to acquire virulence and antibiotic-resistant genes located on mobile genetic elements such as genomic islands, prophages, pathogenicity islands, and the staphylococcal chromosomal cassette mec (SCCmec) by horizontal gene transfer from other staphylococci. The potential virulence of a S. aureus strain is often determined by comparing its pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing profiles to that of known epidemic or virulent clones and by PCR of the toxin genes. Whole-genome mapping (formerly optical mapping), which is a high-resolution ordered restriction mapping of a bacterial genome, is a relatively new genomic tool that allows comparative analysis across entire bacterial genomes to identify regions of genomic similarities and dissimilarities, including small and large insertions and deletions. We explored whether whole-genome maps (WGMs) of methicillin-resistant S. aureus (MRSA) could be used to predict the presence of methicillin resistance, SCCmec type, and Panton-Valentine leukocidin (PVL)-producing genes on an S. aureus genome. We determined the WGMs of 47 diverse clinical isolates of S. aureus, including well-characterized reference MRSA strains, and annotated the signature restriction pattern in SCCmec types, arginine catabolic mobile element (ACME), and PVL-carrying prophage, PhiSa2 or PhiSa2-like regions on the genome. WGMs of these isolates accurately characterized them as MRSA or methicillin-sensitive S. aureus based on the presence or absence of the SCCmec motif, ACME and the unique signature pattern for the prophage insertion that harbored the PVL genes. Susceptibility to methicillin resistance and the presence of mecA, SCCmec types, and PVL genes were confirmed by PCR. A WGM clustering approach was further able to discriminate isolates within the same PFGE clonal group. These results showed that WGMs could be used not only to genotype S. aureus but also to identify genetic motifs in MRSA that may predict virulence.

Use of optical mapping to sort uropathogenic Escherichia coli strains into distinct subgroups.

Optical maps were generated for 33 uropathogenic Escherichia coli (UPEC) isolates. For individual genomes, the NcoI restriction fragments aligned into a unique chromosome map for each individual isolate, which was then compared with the in silico restriction maps of all of the sequenced E. coli and Shigella strains. All of the UPEC isolates clustered separately from the Shigella strains as well as the laboratory and enterohaemorrhagic E. coli strains. Moreover, the individual strains appeared to cluster into distinct subgroups based on the dendrogram analyses. Phylogenetic grouping of these 33 strains showed that 32/33 were the B2 subgroup and 1/33 was subgroup A. To further characterize the similarities and differences among the 33 isolates, pathogenicity island (PAI), haemolysin and virulence gene comparisons were performed. A strong correlation was observed between individual subgroups and virulence factor genes as well as haemolysis activity. Furthermore, there was considerable conservation of sequenced-strain PAIs in the specific subgroups. Strains with different antibiotic-resistance patterns also appeared to sort into separate subgroups. Thus, the optical maps distinguished the UPEC strains from other E. coli strains and further subdivided the strains into distinct subgroups. This optical mapping procedure holds promise as an alternative way to subgroup all E. coli strains, including those involved in infections outside of the intestinal tract and epidemic strains with distinct patterns of antibiotic resistance.

Branched-chain amino acids are required for the survival and virulence of Actinobacillus pleuropneumoniae in swine.

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 micromol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM-BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

Healthcare Antibiotic Resistance Prevalence - DC (HARP-DC): A Regional Prevalence Assessment of Carbapenem-Resistant Enterobacteriaceae (CRE) in Healthcare Facilities in Washington, District of Columbia.

OBJECTIVE Carbapenem-resistant Enterobacteriaceae (CRE) are a significant clinical and public health concern. Understanding the distribution of CRE colonization and developing a coordinated approach are key components of control efforts. The prevalence of CRE in the District of Columbia is unknown. We sought to determine the CRE colonization prevalence within healthcare facilities (HCFs) in the District of Columbia using a collaborative, regional approach. DESIGN Point-prevalence study. SETTING This study included 16 HCFs in the District of Columbia: all 8 acute-care hospitals (ACHs), 5 of 19 skilled nursing facilities, 2 (both) long-term acute-care facilities, and 1 (the sole) inpatient rehabilitation facility. PATIENTS Inpatients on all units excluding psychiatry and obstetrics-gynecology. METHODS CRE identification was performed on perianal swab samples using real-time polymerase chain reaction, culture, and antimicrobial susceptibility testing (AST). Prevalence was calculated by facility and unit type as the number of patients with a positive result divided by the total number tested. Prevalence ratios were compared using the Poisson distribution. RESULTS Of 1,022 completed tests, 53 samples tested positive for CRE, yielding a prevalence of 5.2% (95% CI, 3.9%-6.8%). Of 726 tests from ACHs, 36 (5.0%; 95% CI, 3.5%-6.9%) were positive. Of 244 tests from long-term-care facilities, 17 (7.0%; 95% CI, 4.1%-11.2%) were positive. The relative prevalence ratios by facility type were 0.9 (95% CI, 0.5-1.5) and 1.5 (95% CI, 0.9-2.6), respectively. No CRE were identified from the inpatient rehabilitation facility. CONCLUSION A baseline CRE prevalence was established, revealing endemicity across healthcare settings in the District of Columbia. Our study establishes a framework for interfacility collaboration to reduce CRE transmission and infection. Infect Control Hosp Epidemiol 2017;38:921-929.

A subset of Actinobacillus pleuropneumoniae in vivo induced promoters respond to branched-chain amino acid limitation.

Actinobacillus pleuropneumoniae is the causative agent of a necrotizing hemorrhagic pleuropneumonia in swine. In this study, we investigate the possibility that the limitation of branched-chain amino acids is a stimulus that A. pleuropneumoniae will encounter during infection and will respond to by up-regulation of genes involved in branched-chain amino acid biosynthesis and virulence. Actinobacillus pleuropneumoniae genetic loci that are specifically induced during infection were screened in vitro for expression in response to limitation of branched-chain amino acids. Of 32 in vivo induced promoter clones screened in vitro, eight were induced on chemically defined medium without isoleucine, leucine and valine as compared to complete chemically defined medium. We identify the genomic context of each clone and discuss its relevance to branched-chain amino acid limitation and virulence. We conclude that limitation of branched-chain amino acids is a cue for expression of a subset in vivo induced genes, including not only genes involved in the biosynthesis of branched-chain amino acids, but also other genes that are induced during infection of the natural host. These results suggest that limitation of branched-chain amino acids may be one of an array of environmental cues responsible for the induction of virulence-associated genes in A. pleuropneumoniae.

Scanning the landscape of genome architecture of non-O1 and non-O139 Vibrio cholerae by whole genome mapping reveals extensive population genetic diversity.

Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, ordered restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae, we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.

Identification of the Actinobacillus pleuropneumoniae leucine-responsive regulatory protein and its involvement in the regulation of in vivo-induced genes.

Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset of A. pleuropneumoniae genes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown in Escherichia coli to regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized that A. pleuropneumoniae contains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of an A. pleuropneumoniae serotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purified A. pleuropneumoniae His6-Lrp binds in vitro to the A. pleuropneumoniae promoter regions for ilvI, antisense cps1AB, lrp, and nqr. A genetically defined A. pleuropneumoniae lrp mutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from the ilvI and antisense cps1AB promoters in wild-type, lrp mutant, and complemented lrp mutant strains indicated that Lrp is required for induction of expression of ilvI under BCAA limitation.