Bordetella trematum bacteraemia secondary to an empyema in an immunocompromised host: A case report and review of the literature.

Introduction: Bordetella trematum infection remains uncommon. More cases of bacteraemia are reported in recent years with the primary infection largely originating from skin and soft tissue sites. Yet, our understanding of its virulence, antibiotic susceptibility profile and treatment is still limited. Case presentation: Case presentation. We report the first case of B. trematum bacteraemia from a left-sided empyema. An 87-year-old female patient with a past medical history of ischaemic heart disease, diabetes mellitus complicated by nephropathy and locally advanced left breast adenocarcinoma presented with fever, productive cough and shortness of breath. The B. trematum isolates from blood and pleural fluid were identified by MALDI-TOF and 16S rRNA sequencing. Ceftriaxone and azithromycin commenced empirically on admission were switched to piperacillin-tazobactam after 2 days due to lack of clinical improvement. Despite a pleurocentesis and 1 week of piperacillin-tazobactam with microbiological clearance in blood, the patient continued to deteriorate. Decision to withdraw treatment was made in view of the patient's prognosis, and the patient succumbed on the fourteenth day of admission. The isolate was susceptible to piperacillin-tazobactam, imipenem and meropenem but had reduced susceptibility or was non-susceptible to cefuroxime, cefotaxime, ceftazidime, cefepime, the aminoglycosides and fluoroquinolones. Conclusion: Invasive B. trematum infection is associated with significant mortality. Consensus for antibiotic treatment remains unclear, with limited susceptibility data to support specific antibiotic use. We expect more clinical cases will surface with improved microbial identification systems, as well as enhanced clinical awareness. Standardized and more robust susceptibility work are needed to provide clear recommendations and establish consensus in treating invasive infections.

A Study of Analytical and Clinical Sensitivity of Aptima SARS-CoV-2 Assay (Hologic) and Proposals of Complementary Tests for SARS-CoV-2 Detection in Low Viral Load Specimens.

Early and accurate detection of SARS-CoV-2 is important for diagnosis and transmission control. The use of high-throughput and automated testing allows laboratories to better deliver diagnostic testing given manpower and resource limitations. We validated the clinical and analytical performance of the Hologic Panther Aptima SARS-CoV-2 assay with an emphasis on detection of specimens with low viral loads. The clinical performance was evaluated using 245 clinical specimens, against a comparator PCR-based laboratory developed test (LDT). The analytical performance was determined by replicate testing of contrived samples in a ten-fold dilution series (CT values 32-42, based on LDT). The Aptima assay had 96.7% overall percent agreement, 100% negative percent agreement and 88.1% positive percent agreement. It was able to consistently detect SARS-CoV-2 in contrived samples with CT = 32 by LDT (calculated 2354 copies/mL). The 95% limit of detection of the Aptima assay was estimated to be at LDT CT = 33 (equivalent to 870 copies/mL). The relative light units (RLU) × 1000 for 52 true positive clinical specimens was 962.2 ± 181.5, and that for the 186 true negative specimens was 264.6 ± 14.3. The Aptima assay was a reliable method with a high overall percent agreement against our comparator LDT. We propose that samples reported as negative by the Aptima assay with RLU > 350 be tested by a secondary method, in order to improve detection of samples with very low viral loads.

Development of a rapid multiplex PCR assay for the detection of common pathogens associated with community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is one of the most common infectious diseases and is a significant cause of mortality and morbidity globally. A microbial cause was not determined in a sizable percentage of patients with CAP; there are increasing data to suggest regional differences in bacterial aetiology. We devised a multiplex real-time PCR assay for detecting four microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Burkholderia pseudomallei) of relevance to CAP infections in Asia. METHODS: Analytical validation was accomplished using bacterial isolates (n=10-33 of each target organism for analytical sensitivity and n=117 for analytical sensitivity) and clinical validation using 58 culture-positive respiratory tract specimens. RESULTS: The qPCR assay exhibited 100% analytical sensitivity and analytical specificity, and 100% clinical sensitivity and 94-100% clinical specificity. The limit of detection and efficiency for the multiplex PCR assay were 3-33 CFU/mL and 93-110%, respectively. The results showed that the PCR-based method had higher sensitivity than traditional culture-based methods. The assay also demonstrated an ability to semiquantify bacterial loads. CONCLUSION: We have devised a reliable laboratory-developed multiplex qPCR assay, with a turnaround time of within one working day, for detection of four clinically important CAP-associated microorganisms in Asia. The availability of a test with improved diagnostic capabilities potentially leads to an informed choice of antibiotic usage and appropriate management of the patient to achieve a better treatment outcome and financial savings.

Prevalence and antibiotic susceptibility of colistin-resistance gene (mcr-1) positive Enterobacteriaceae in stool specimens of patients attending a tertiary care hospital in Singapore.

OBJECTIVES: The aim of this study was to determine the prevalence of the colistin-resistance gene (mcr-1) and the antibiotic-susceptibility profile of mcr-1 positive, colistin-resistant isolates in stool specimens of patients attending a tertiary care hospital in Singapore. METHODS: 201 diarrheal stool specimens of patients attending the Changi General Hospital between May to August 2017 were collected and screened for the presence of mcr-1 by culture and molecular methods. Antibiotic-susceptibility profile of mcr-1 positive isolates was determined using the polymyxin B and colistin E-tests and the VITEK 2 system. RESULTS: We observed an unexpectedly high prevalence of mcr-1 in patients attending a tertiary care hospital in Singapore, i.e 6.0% and 8.0% estimated by stool culture and direct stool PCR, respectively. The mcr-1 gene was detected predominantly in Escherichia coli. Antibiotic-susceptibility testing on 12 mcr-1 positive Enterobacteriaceae isolates revealed variable susceptibility profiles with no detection of carbapenem-resistant Enterobacteriaceae. CONCLUSIONS: This is the first report of the prevalence of human faecal carriage of mcr-1 in Singapore. Our findings highlight the potential risk of mcr-1 spread among our patient cohort. The mcr-1 gene detection combined with the detection of other resistance gene targets of clinical importance is recommended to pre-empt the spread mcr-1 in our patients.

Retrospective genome-wide comparisons of Salmonella enterica serovar Enteritidis from suspected outbreaks in Singapore.

The number of salmonellosis cases in Singapore has increased over the years. Salmonella enterica serovar Enteritidis has always been the most predominant serovar in the last five years. The National Public Health Laboratory assisted outbreak investigations by performing multilocus variable number tandem repeat analysis (MLVA) on isolates that were collected at the time of the investigations. Isolates were defined as belonging to a particular cluster if they had identical MLVA patterns. Whilst MLVA has been instrumental in outbreak investigations, it may not be useful when outbreaks are caused by an endemic MLVA type. In this study, we analysed 67 isolates from 12 suspected outbreaks with known epidemiological links to explore the use of next-generation sequencing (NGS) for defining outbreaks. We found that NGS can confidently group isolates into their respective outbreaks. The isolates from each suspected outbreak were closely related and differed by a maximum of 3 single nucleotide polymorphisms (SNPs). They were also clearly separated from isolates that belonged to different suspected outbreaks. This study provides an important insight and further evidence on the value of NGS for routine surveillance and outbreak detection of S. Enteritidis.

Draft Genome Sequence of Singapore Klebsiella pneumoniae subsp. pneumoniae Isolate DS32358_14, Which Contains the Carbapenemase Gene blaVIM-1.

We sequenced the first blaVIM-1-positive Klebsiella pneumoniae strain isolated in Singapore. The isolate belongs to multilocus sequence type 2542 (ST2542), and blaVIM-1 was the first gene in an integron that also contained aacA4, aphA15, aadA1, catB2, qacEdelta1, and sul1.

Putative Integrative Mobile Elements That Exploit the Xer Recombination Machinery Carrying blaIMI-Type Carbapenemase Genes in Enterobacter cloacae Complex Isolates in Singapore.

Whole-genome sequencing was performed on 16 isolates of the carbapenemase-producing Enterobacter cloacae complex to determine the flanking regions of blaIMI-type genes. Phylogenetic analysis of multilocus sequence typing (MLST) targets separated the isolates into 4 clusters. The blaIMI-type genes were all found on Xer-dependent integrative mobile elements (IMEX). The IMEX elements of 5 isolates were similar to those described in Canada, while the remainder were novel. Five isolates had IMEX elements lacking a resolvase and recombinase.

Colistin and Polymyxin B Susceptibility Testing for Carbapenem-Resistant and mcr-Positive Enterobacteriaceae: Comparison of Sensititre, MicroScan, Vitek 2, and Etest with Broth Microdilution.

Colistin and polymyxin B remain part of the last line of antibiotics for multidrug-resistant Gram-negative bacteria, such as carbapenem-resistant Enterobacteriaceae Current joint EUCAST-CLSI recommendations are for broth microdilution (BMD) to be performed for MIC testing of colistin. Commercial susceptibility testing methods were evaluated and compared against the reference BMD, using a susceptibility breakpoint of ≤2 mg/liter for both colistin and polymyxin B. Seventy-six Enterobacteriaceae were included, of which 21 were mcr-1 positive (18 Escherichia coli isolates, 2 Klebsiella pneumoniae isolates, and 1 Enterobacter aerogenes isolate). Rates of essential agreement (EA) of colistin test results between BMD and Vitek 2, Sensititre, and Etest were 93.4%, 89.5%, and 75.0%, respectively. Rates of EA of polymyxin B test results between BMD and Vitek 2, Sensititre, and Etest were 96.1%, 96.1%, and 48.7%, respectively. A positive MIC correlation with a categorical agreement of >90% was achieved for Sensititre (colistin Spearman's ρ = 0.863, and polymyxin B Spearman's ρ = 0.877) and Vitek 2 (polymyxin B [only] Spearman's ρ = 0.8917). Although a positive MIC correlation (Spearman's ρ = 0.873) with the reference method was achieved for colistin testing with Vitek 2, categorical agreement was <90%, with very major error rates of 36%. Correlation with the Etest MIC was lower, with very major error rates of 12% (colistin) and 26.1% (polymyxin B). MicroScan (colistin) categorical agreement was 88.2%, with a very major error rate of 4%. Colistin MICs for 15 of the 21 mcr-1-positive isolates were >2 mg/liter, and polymyxin MICs for 17 of them were >2 mg/liter by broth microdilution. The use of a lower breakpoint of ≤1 mg/liter further improves detection of mcr-1 for all testing methods. However, further data on the correlation between MICs and clinical outcome are required to determine the most suitable breakpoint to guide clinical management.

Development and evaluation of a multiplex real-time PCR for the detection of IMP, VIM, and OXA-23 carbapenemase gene families on the BD MAX open system.

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of clinically prevalent IMP, VIM, and OXA-23 gene families. The assay was designed to work on the BD MAX open platform which is a fully automated system for all PCR processes including sample extraction to PCR resulting. A total of 107 well-characterized carbapenem resistant Enterobacteriaceae were evaluated and the results were 100% concordant with the reference test isolates. This assay will serve to complement PCR screens that detect the major carbapenemase families of NDM, KPC, and OXA-48-like.

Outbreak of New Delhi metallo-ß-lactamase-1-producing Enterobacter cloacae in an acute care hospital general ward in Singapore.

BACKGROUND: Autochthonous infections with New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae have been reported in Singapore since 2011, but occurrences of nosocomial transmission have not. We report an outbreak of NDM-1-producing Enterobacter cloacae among adults admitted to an acute hospital's general ward. METHODS: On detecting the index case with a culture specimen positive for NDM-1-producing E cloacae, active case finding was conducted by screening all possible patient contacts. On-site ward assessment was performed, and electronic patient medical records were reviewed to conduct a case-control study to identify factors associated with colonization. RESULTS: Of 55 screened patient contacts, 3 further cases were detected, with isolates genetically related to the index case. None of these 4 cases was housed within the same cubicle. However, 3 were managed by the same medical team. Being managed by this team was positively associated with being a case (adjusted odds ratio = 15.64; 95% confidence interval, 0.91-270.27; P = .06) after adjusting for age, sex, Charlson comorbidity index score, and recent antibiotic use. CONCLUSION: Our report suggests nosocomial transmission of NDM-1-producing E cloacae occurred via health care staff. Improvements in infection control measures, especially pertaining to staff hand hygiene practices and ward staffing, are needed to reduce the spread of highly resistant pathogens, such as NDM-1-producing Enterobacteriaceae.

Performance assessment of the BD MAX Cdiff assay in comparison to Xpert C. difficile assay in a setting with very low prevalence of toxigenic Clostridium difficile PCR ribotype 027.

In a clinical setting with low prevalence of 'epidemic' PCR ribotype 027, the BD MAX Cdiff assay was found to be a suitable alternative to the Xpert C. difficile assay for the detection of toxigenic Clostridium difficile in samples which are reflex PCR tested after obtaining a discrepant immunoassay result. There was no significant difference between the sensitivities and specificities of both commercial molecular assays.

Surveillance trends of carbapenem-resistant Enterobacteriaceae from Singapore, 2010-2013.

Trends in carbapenem-resistant Enterobacteriaceae (CRE) collected from hospitals nationwide in Singapore over 3 years are presented. Hospital isolates with imipenem or meropenem minimum inhibitory concentrations (MICs) of >1mg/L were sent to the National Public Health Laboratory for further investigation. A total of 400 CRE were submitted, 227 (56.8%) of which carried a carbapenemase gene. blaNDM was the most common (130/400; 32.5%), followed by blaOXA-48-like (blaOXA-48, -181, -232) (55/400; 13.8%). Interestingly, four isolates bearing dual carbapenemase genes were also detected. KPC- and OXA-48-like-producing Klebsiella pneumoniae were fingerprinted by DiversiLab® rep-PCR. Locally, KPC producers do not appear to have clonal dissemination. In contrast, OXA-48-like producers were found to have a greater degree of clustering than KPC producers.

Enterobacter cloacae producing an uncommon class A carbapenemase, IMI-1, from Singapore.

In this study, we describe the characterization of an infrequently encountered class A carbapenemase, IMI-1, from a clinical Enterobacter cloacae isolate. The isolate had high levels of resistance to carbapenems but retained susceptibility to expanded-spectrum cephalosporins. The blaIMI-1 gene was chromosomally encoded. Detection of the IMI-1 producer highlights the diversity of carbapenemases in a local clinical setting.

Finegoldia magna, an early post-operative cause of infectious endocarditis: report of two cases and review of the literature.

We report two cases of infectious endocarditis (IE) on prosthetic valves caused by Finegoldia magna. The diagnosis was obtained by detection of the bacterium in valvular biopsies using 16S rRNA PCR amplication and sequencing, and prolonged culture. Five other cases were previously published in the literature. Following analysis of these seven cases, F. magna endocarditis presented as a subacute endocarditis, developing early (60 days) following valvular replacement (85%), with an elevated mortality (28%). Our report highlights the potential role of F. magna in early post-surgical endocarditis on prosthetic valves.

Regulation of whole bacterial pathogen transcription within infected hosts.

DNA microarrays are a powerful and promising approach to gain a detailed understanding of the bacterial response and the molecular cross-talk that can occur as a consequence of host-pathogen interactions. However, published studies mainly describe the host response to infection. Analysis of bacterial gene regulation in the course of infection has confronted many challenges. This review summarizes the different strategies used over the last few years to investigate, at the genomic scale, and using microarrays, the alterations in the bacterial transcriptome in response to interactions with host cells. Thirty-seven studies involving 19 different bacterial pathogens were compiled and analyzed. Our in silico comparison of the transcription profiles of bacteria grown in broth or in contact with eukaryotic cells revealed some features commonly observed when bacteria interact with host cells, including stringent response and cell surface remodeling.

Development of a method for recovering rickettsial RNA from infected cells to analyze gene expression profiling of obligate intracellular bacteria.

The Rickettsia genus is composed of Gram-negative bacteria responsible for Typhus and spotted fevers. Because of the limitations imposed by their obligate intracellular location, the molecular mechanisms responsible for their pathogenicity remain poorly understood. Several rickettsial genomes are now available, thus providing the foundation for a new era of post-genomic research. Here, using Rickettsia conorii as model, we developed a suitable method for microarray-based transcriptome analysis of rickettsiae. Total RNA was extracted from infected Vero cells using a protocol preserving its integrity, as observed by Bioanalyzer (Agilent) profiles. By a subtractive hybridization method, the samples were subsequently depleted of eukaryotic RNA that represents up to 90% of the whole extract and that hampers fluorochrome labeling of rickettsial nucleic acids. To obtain the amount of material required for microarray hybridization, the bacterial RNA was then amplified using random primers. Hybridizations were carried out on microarrays specific for R. conorii but containing a limited number of selected targets. Our results show that this method yielded reproducible signals. Transcriptional changes observed following exposure of R. conorii to a nutrient stress were verified by real-time quantitative PCR and by quantitative reverse transcription PCR starting from amplified cDNA and total RNA as templates, respectively. We conclude that this approach has great potential for the study of mechanisms behind the virulence and intracellular survival of members of the genus Rickettsia.

Comparative genomic analysis of Tropheryma whipplei strains reveals that diversity among clinical isolates is mainly related to the WiSP proteins.

BACKGROUND: The aim of this study was to analyze the genomic diversity of several Tropheryma whipplei strains by microarray-based comparative genomic hybridization. Fifteen clinical isolates originating from biopsy samples recovered from different countries were compared with the T. whipplei Twist strain. For each isolate, the genes were defined as either present or absent/divergent using the GACK analysis software. Genomic changes were then further characterized by PCR and sequencing. RESULTS: The results revealed a limited genetic variation among the T. whipplei isolates, with at most 2.24% of the probes exhibiting differential hybridization against the Twist strain. The main variation was found in genes encoding the WiSP membrane protein family. This work also demonstrated a 19.2 kb-pair deletion within the T. whipplei DIG15 strain. This deletion occurs in the same region as the previously described large genomic rearrangement between Twist and TW08/27. Thus, this can be considered as a major hot-spot for intra-specific T. whipplei differentiation. Analysis of this deleted region confirmed the role of WND domains in generating T. whipplei diversity. CONCLUSION: This work provides the first comprehensive genomic comparison of several T. whipplei isolates. It reveals that clinical isolates originating from various geographic and biological sources exhibit a high conservation rate, indicating that T. whipplei rarely interacts with exogenous DNA. Remarkably, frequent inter-strain variations were dicovered that affected members of the WiSP family.