Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniae and E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

Estimated glomerular filtration rate as a tool for early identification of patients with insufficient exposure to beta-lactam antibiotics in intensive care units.

BACKGROUND: Only about 50% of intensive care unit (ICU) patients reach a free trough concentration above MIC (100% fT > MIC) of beta-lactam antibiotics. Although dose adjustments based on therapeutic drug monitoring (TDM) could be beneficial, TDM is not widely available. We investigated serum creatinine-based estimated GFR (eGFR) as a rapid screening tool to identify ICU patients at risk of insufficient exposure. METHOD: Ninety-three adult patients admitted to four ICUs in southeast Sweden treated with piperacillin/tazobactam, meropenem, or cefotaxime were included. Beta-lactam trough concentrations were measured. The concentration target was set to 100% fT > MICECOFF (2, 4, and 16 mg/L based on calculated free levels for meropenem, cefotaxime, and piperacillin, respectively). eGFR was primarily determined via Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) and compared to three other eGFR equations. Data was analysed using logistic regression and receiver operative characteristic (ROC) curves. RESULTS: With intermittent standard dosing, insufficient exposure was common in patients with a relative eGFR ≥48mL/min/1.73m2 [85%, (45/53)], particularly when treated with cefotaxime [96%, (24/25)]. This eGFR cut-off had a sensitivity of 92% and specificity of 82% (AUC 0.871, p < 0.001) in identifying insufficient exposure. In contrast, patients with eGFR <48mL/min/1.73m2 had high target attainment [90%, (36/40)] with a wide variability in drug exposure. There was no difference between the four eGFR equations (AUC 0.866-0.872, cut-offs 44-51 ml/min/1.73m2). CONCLUSION: Serum creatinine-based eGFR is a simple and widely available surrogate marker with potential for early identification of ICU patients at risk of insufficient exposure to piperacillin, meropenem, and cefotaxime.

Dissemination of carbapenemase-producing Enterobacterales through wastewater and gulls at a wastewater treatment plant in Sweden.

Here we report the detection of carbapenemase-producing Enterobacterales (CPE) isolated from Swedish wastewater and gull faeces. CPE have not been detected in samples from animals in Sweden preceding this report. Sampling of wastewater treatment plant (WWTP) inlet and outlet, sedimentation basins, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions during May to September 2021. Following broth enrichment, selective screening of putative CPE was performed on mSuperCarba™ (CHROMagar). Species identification was done with MALDI-TOF. Antimicrobial susceptibility testing was performed according to EUCAST. In total, seventeen CPE were verified by genome sequencing carrying blaGES-5, blaIMI-3, blaOXA-181 or blaOXA-244. The blaGES-5 was carried on IncP plasmids in four different species; Escherichia coli ST10 isolated from WWTP outlet, Raoultella ornithinolytica isolated from WWTP inlet, outlet and sedimentation basins as well as gull faeces collected at the WWTP and Klebsiella spp. isolates from WWTP inlet and outlet. The genetic environment surrounding blaGES-5 was similar in two Citrobacter freundii causing human infections. The blaIMI-3 was carried on IncFII(Yp) plasmids in four Enterobacter ludwigii, isolated from WWTP outlet and gull faeces collected at a recreational city park 2 km from the WWTP. The blaOXA-181 was located on a COLKP3 plasmid found in an E. coli, while blaOXA-244 was chromosomally located in an E. coli ST10, both isolated from WWTP inlet. Phylogenetic analysis of R. ornithinolytica and E. ludwigii isolates indicate that the gulls carried strains related to those identified in the WWTP samples. The results thus add to the increasing evidence of WWTPs as anthropogenic reservoirs for mobile genetic elements with antibiotic-resistance functionality. Such environments could profoundly impact the dissemination and spread of such genetic elements via for example aquatic birds, thereby warranting further study and surveillance.

Exchange of Carbapenem-Resistant Escherichia coli Sequence Type 38 Intercontinentally and among Wild Bird, Human, and Environmental Niches.

Carbapenem-resistant Enterobacteriaceae (CRE) are a global threat to human health and are increasingly being isolated from nonclinical settings. OXA-48-producing Escherichia coli sequence type 38 (ST38) is the most frequently reported CRE type in wild birds and has been detected in gulls or storks in North America, Europe, Asia, and Africa. The epidemiology and evolution of CRE in wildlife and human niches, however, remains unclear. We compared wild bird origin E. coli ST38 genome sequences generated by our research group and publicly available genomic data derived from other hosts and environments to (i) understand the frequency of intercontinental dispersal of E. coli ST38 clones isolated from wild birds, (ii) more thoroughly measure the genomic relatedness of carbapenem-resistant isolates from gulls sampled in Turkey and Alaska, USA, using long-read whole-genome sequencing and assess the spatial dissemination of this clone among different hosts, and (iii) determine whether ST38 isolates from humans, environmental water, and wild birds have different core or accessory genomes (e.g., antimicrobial resistance genes, virulence genes, plasmids) which might elucidate bacterial or gene exchange among niches. Our results suggest that E. coli ST38 strains, including those resistant to carbapenems, are exchanged between humans and wild birds, rather than separately maintained populations within each niche. Furthermore, despite close genetic similarity among OXA-48-producing E. coli ST38 clones from gulls in Alaska and Turkey, intercontinental dispersal of ST38 clones among wild birds is uncommon. Interventions to mitigate the dissemination of antimicrobial resistance throughout the environment (e.g., as exemplified by the acquisition of carbapenem resistance by birds) may be warranted. IMPORTANCE Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene blaOXA-48. This is the most frequently reported carbapenem-resistant clone in wild birds, though it was unclear if it circulated within wild bird populations or was exchanged among other niches. The results from this study suggest that E. coli ST38 strains, including those resistant to carbapenems, are frequently exchanged among wild birds, humans, and the environment. Carbapenem-resistant E. coli ST38 clones in wild birds are likely acquired from the local environment and do not constitute an independent dissemination pathway within wild bird populations. Management actions aimed at preventing the environmental dissemination and acquisition of antimicrobial resistance by wild birds may be warranted.

High prevalence of blaCTX-M-15 type extended-spectrum beta-lactamases in Gambian hooded vultures (Necrosyrtes monachus): A threatened species with substantial human interaction.

One hundred fecal samples from hooded vultures in the Gambia (Banjul area) were investigated for the presence of bacteria with extended-spectrum cephalosporin- (ESBL/AmpC), carbapenemases, and colistin resistance. No Enterobacteriales carrying carbapenemases or resistance against colistin were detected. Fifty-four ESBL-producing Escherichia coli and five ESBL-producing Klebsiella pneumoniae isolates were identified in 52 of the samples, of which 52 E. coli and 4 K. pneumoniae yielded passed sequencing results. Fifty of the E. coli had ESBL phenotype and genotype harboring blaCTX-M genes, of which 88.5% (n = 46) were the blaCTX-M-15 gene, commonly found on the African continent. Furthermore, the genetic context around blaCTX-M-15 was similar between isolates, being colocalized with ISKpn19. In contrast, cgMLST analysis of the E. coli harboring ESBL genes revealed a genetic distribution over a large fraction of the currently known existing E. coli populations in the Gambia. Hooded vultures in the Gambia thus have a high ESBL E. coli-prevalence (>50%) with low diversity regarding key resistance genes. Furthermore, given the urban presence and frequent interactions between hooded vultures and humans, data from this study implies hooded vultures as potential vectors contributing to the further dissemination of antibiotic-resistance genes.

Environmental antimicrobial resistance gene detection from wild bird habitats using two methods: A commercially available culture-independent qPCR assay and culture of indicator bacteria followed by whole-genome sequencing.

OBJECTIVES: A variety of methods have been developed to detect antimicrobial resistance (AMR) in different environments to better understand the evolution and dissemination of this public health threat. Comparisons of results generated using different AMR detection methods, such as quantitative PCR (qPCR) and whole-genome sequencing (WGS), are often imperfect, and few studies have analysed samples in parallel to evaluate differences. In this study, we compared bacterial culture and WGS to a culture-independent commercially available qPCR assay to evaluate the concordance between methods and the utility of each in answering research questions regarding the presence and epidemiology of AMR in wild bird habitats. METHODS: We first assessed AMR gene detection using qPCR in 45 bacterial isolates from which we had existing WGS data. We then analysed 52 wild bird faecal samples and 9 spatiotemporally collected water samples using culture-independent qPCR and WGS of phenotypically resistant indicator bacterial isolates. RESULTS: Overall concordance was strong between qPCR and WGS of bacterial isolates, although concordance differed among antibiotic classes. Analysis of wild bird faecal and water samples revealed that more samples were determined to be positive for AMR via qPCR than via culture and WGS of bacterial isolates, although qPCR did not detect AMR genes in two samples from which phenotypically resistant isolates were found. CONCLUSIONS: Both qPCR and culture followed by sequencing may be effective approaches for characterising AMR genes harboured by wild birds, although data streams produced using these different tools may have advantages and disadvantages that should be considered given the application and sample matrix.

Genomically diverse carbapenem resistant Enterobacteriaceae from wild birds provide insight into global patterns of spatiotemporal dissemination.

Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenically-associated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

Simultaneous Measurement of 11 Antibiotics for Use in the Intensive Care Unit by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Recent studies indicate that a high proportion of patients in the intensive care unit fail to attain adequate antibiotic levels. Thus, there is a need to monitor the antibiotic concentration to ensure effective treatment. In this article, the authors aimed to develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of antimicrobials to assess individualized therapeutic drug monitoring. METHODS: A UHPLC-MS/MS method with 11 antibiotics (ciprofloxacin, moxifloxacin, benzylpenicillin, levofloxacin, linezolid, rifampicin, meropenem, cloxacillin, cefotaxime, clindamycin, and piperacillin) was developed. Chromatographic separation was performed using a Kinetex Biphenyl reversed-phase column, with gradient elution using 0.1% formic acid and methanol with 0.1% formic acid. Sample preparation was performed using methanol protein precipitation. The total run time was 5 minutes. RESULTS: For all analytes, the interassay inaccuracies for calibrators were ≤5%. The interday inaccuracies for the quality controls (QCs) were ≤5% for all analytes. The interassay precision for calibration standards ranged between 1.42% and 6.11%. The interassay imprecision for QCs of all antibiotics and concentrations ranged between 3.60% and 16.1%. Interassay inaccuracy and imprecision for the QCs and calibration standards were ≤15% for all drugs, except benzylpenicillin. CONCLUSIONS: A rapid UHPLC-MS/MS method was developed for the simultaneous quantification of 11 different antibiotics. Minimal sample preparation was required to ensure a rapid turnaround time. The method was applied to clinical samples collected from 4 intensive care units.

Evidence for continental-scale dispersal of antimicrobial resistant bacteria by landfill-foraging gulls.

Anthropogenic inputs into the environment may serve as sources of antimicrobial resistant bacteria and alter the ecology and population dynamics of synanthropic wild animals by providing supplemental forage. In this study, we used a combination of phenotypic and genomic approaches to characterize antimicrobial resistant indicator bacteria, animal telemetry to describe host movement patterns, and a novel modeling approach to combine information from these diverse data streams to investigate the acquisition and long-distance dispersal of antimicrobial resistant bacteria by landfill-foraging gulls. Our results provide evidence that gulls acquire antimicrobial resistant bacteria from anthropogenic sources, which they may subsequently disperse across and between continents via migratory movements. Furthermore, we introduce a flexible modeling framework to estimate the relative dispersal risk of antimicrobial resistant bacteria in western North America and adjacent areas within East Asia, which may be adapted to provide information on the risk of dissemination of other organisms and pathogens maintained by wildlife through space and time.

Celiac disease and complement activation in response to Streptococcus pneumoniae.

Individuals with celiac disease (CD) are at increased risk of invasive pneumococcal disease (IPD). The aim of this study was to explore whether the complement response to Streptococcus pneumoniae differed according to CD status, and could serve as an explanation for the excess risk of IPD in CD. Twenty-two children with CD and 18 controls, born 1999-2008, were included at Kalmar County Hospital, Sweden. The degree of complement activation was evaluated by comparing levels of activation products C3a and sC5b-9 in plasma incubated for 30 min with Streptococcus pneumoniae and in non-incubated plasma. Complement analyses were performed with enzyme-linked immunosorbent assay (ELISA). Pneumococcal stimulation caused a statistically significant increase in C3a as well as sC5b-9 in both children with CD and controls but there was no difference in response between the groups. After incubation, C3a increased on average 4.6 times and sC5b-9 22 times in both the CD and the control group (p = 0.497 and p = 0.724 respectively).Conclusion: Complement response to Streptococcus pneumoniae seems to be similar in children with and without CD and is thus unlikely to contribute to the increased susceptibility to invasive pneumococcal disease in CD.What is Known:• An excess risk of pneumococcal infections has been demonstrated in individuals with celiac disease.• Infectious complications can depend on hyposplenism but alternative mechanisms are sparsely examined.What is New:• Complement activation in response to Streptococcus pneumoniae was examined in children with and without celiac disease but no differences could be demonstrated.

Repeated Detection of Carbapenemase-Producing Escherichia coli in Gulls Inhabiting Alaska.

Here, we report the first detection of carbapenemase-producing Escherichia coli in Alaska and in wildlife in the United States. Wild bird (gull) feces sampled at three locations in Southcentral Alaska yielded isolates that harbored plasmid-encoded blaKPC-2 or chromosomally encoded blaOXA-48 and genes associated with antimicrobial resistance to up to eight antibiotic classes.

Early emergence of mcr-1-positive Enterobacteriaceae in gulls from Spain and Portugal.

We tested extended-spectrum ß-lactamase producing bacteria from wild gulls (Larus spp.) sampled in 2009 for the presence of mcr-1. We report the detection of mcr-1 and describe genome characteristics of four Escherichia coli and one Klebsiella pneumoniae isolate from Spain and Portugal that also exhibited colistin resistance. Results represent the earliest evidence for colistin-resistant bacteria in European wildlife.

Satellite tracking of gulls and genomic characterization of faecal bacteria reveals environmentally mediated acquisition and dispersal of antimicrobial-resistant Escherichia coli on the Kenai Peninsula, Alaska.

Gulls (Larus spp.) have frequently been reported to carry Escherichia coli exhibiting antimicrobial resistance (AMR E. coli); however, the pathways governing the acquisition and dispersal of such bacteria are not well described. We equipped 17 landfill-foraging gulls with satellite transmitters and collected gull faecal samples longitudinally from four locations on the Kenai Peninsula, Alaska to assess: (a) gull attendance and transitions between sites, (b) spatiotemporal prevalence of faecally shed AMR E. coli, and (c) genomic relatedness of AMR E. coli isolates among sites. We also sampled Pacific salmon (Oncorhynchus spp.) harvested as part of personal-use dipnet fisheries at two sites to assess potential contamination with AMR E. coli. Among our study sites, marked gulls most commonly occupied the lower Kenai River (61% of site locations) followed by the Soldotna landfill (11%), lower Kasilof River (5%) and upper Kenai River (<1%). Gulls primarily moved between the Soldotna landfill and the lower Kenai River (94% of transitions among sites), which were also the two locations with the highest prevalence of AMR E. coli. There was relatively high spatial and temporal variability in AMR E. coli prevalence in gull faeces and there was no evidence of contamination on salmon harvested in personal-use fisheries. We identified E. coli sequence types and AMR genes of clinical importance, with some isolates possessing genes associated with resistance to as many as eight antibiotic classes. Our findings suggest that gulls acquire AMR E. coli at habitats with anthropogenic inputs and subsequent movements may represent pathways through which AMR is dispersed.

Two-peaked increase of serum myosin heavy chain-α after triathlon suggests heart muscle cell death.

OBJECTIVE: It has been suggested that the mechanism behind cardiac troponin elevation after strenuous exercise is passage through a cell membrane with changed permeability rather than myocardial cell death. We hypothesised that an increase of cardiac specific myosin heavy chain-alpha (MHC-α; 224 kDa compared with cardiac troponin T's (cTnT) 37 kDa) could hardly be explained by passage through a cell membrane. METHODS: Blood samples were collected from 56 athletes (15 female, age 42.5±9.7, range 24-70 years) before, directly after and on days 1-8 after an Ironman. Biomarkers (C reactive protein (CRP), cTnT, creatine kinase (CK), MHC-α, myoglobin (MG), creatinine (C) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) were measured. RESULTS: The course of MHC-α concentration (µg/L) was 1.33±0.53 (before), 2.57±0.78 (directly after), 1.51±0.53 (day 1), 2.74±0.55 (day 4) and 1.83±0.76 (day 6). Other biomarkers showed a one-peaked increase with maximal values either directly after the race or at day 1: cTnT 76 ± 80 ng/L (12-440; reference<15), NT-proBNP 776±684 ng/L (92-4700; ref.<300), CK 68±55 µkat/L (5-280; ref.<1.9), MG 2088±2350 µg/L (130-17 000; ref.<72) and creatinine 100±20 µmol/L (74-161; ref.<100), CRP 49±23 mg/L (15-119; ref.<5). CONCLUSION: MHC-α exhibited a two-peaked increase which could represent a first release from the cytosolic pool and later from cell necrosis. This is the first investigation of MHC-α plasma concentration after exercise.

Acquisition and dissemination of cephalosporin-resistant E. coli in migratory birds sampled at an Alaska landfill as inferred through genomic analysis.

Antimicrobial resistance (AMR) in bacterial pathogens threatens global health, though the spread of AMR bacteria and AMR genes between humans, animals, and the environment is still largely unknown. Here, we investigated the role of wild birds in the epidemiology of AMR Escherichia coli. Using next-generation sequencing, we characterized cephalosporin-resistant E. coli cultured from sympatric gulls and bald eagles inhabiting a landfill habitat in Alaska to identify genetic determinants conferring AMR, explore potential transmission pathways of AMR bacteria and genes at this site, and investigate how their genetic diversity compares to isolates reported in other taxa. We found genetically diverse E. coli isolates with sequence types previously associated with human infections and resistance genes of clinical importance, including blaCTX-M and blaCMY. Identical resistance profiles were observed in genetically unrelated E. coli isolates from both gulls and bald eagles. Conversely, isolates with indistinguishable core-genomes were found to have different resistance profiles. Our findings support complex epidemiological interactions including bacterial strain sharing between gulls and bald eagles and horizontal gene transfer among E. coli harboured by birds. Results suggest that landfills may serve as a source for AMR acquisition and/or maintenance, including bacterial sequence types and AMR genes relevant to human health.

Epidemiological characterization of a nosocomial outbreak of extended spectrum ß-lactamase Escherichia coli ST-131 confirms the clinical value of core genome multilocus sequence typing.

Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST-131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST-131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability.

Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages.

BACKGROUND: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. METHOD: Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. RESULTS: When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. CONCLUSION: In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.

A microbiological method for determining serum levels of broad spectrum ß-lactam antibiotics in critically ill patients.

BACKGROUND: Recent studies show that suboptimal blood levels of ß-lactam antibiotics are present in intensive care unit (ICU) patients. A common reference method for assessing drug concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is highly accurate but rarely available outside reference centres. Thus, our aim was to develop a microbiological method for monitoring ß-lactam antibiotic serum levels which could be used at any hospital with a microbiological laboratory. METHODS: The method was developed as a 96-well broth microdilution format to assess the concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient serum containing antibiotics were diluted in suspensions of bacteria with known minimal inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing the MIC with the dilution factor at which the serum inhibited growth of the bacterial suspension. Serum (n=88) from ICU patients at four hospitals in south-east Sweden were analysed and compared to LC-MS analysis. RESULTS: The overall accuracy and precision for spiked samples and patient samples was within the pre-set target of ±20.0% for all drugs. There was a significant correlation between the microbiological assay and LC-MS for the patient samples (CTX: r=0.86, n=31; MER: r=0.96, n=11; PIP: r=0.88, n=39) and the agreement around the clinical cut-off for CTX (4.0mg/l), MER (2.0mg/l) and PIP (16.0mg/l) was 90%, 100% and 87%, respectively. CONCLUSION: The microbiological method has a performance for determination of serum levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive method applicable in any microbiology laboratory.

Evaluation of high-resolution melting curve analysis of ligation-mediated real-time PCR, a rapid method for epidemiological typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) pathogens.

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks.

Rapid identification of pneumococci, enterococci, beta-haemolytic streptococci and S. aureus from positive blood cultures enabling early reports.

BACKGROUND: The aim of this study was to evaluate diagnostic tests in order to introduce a diagnostic strategy to identify the most common gram-positive bacteria (pneumococci, enterococci, ß-haemolytic streptococci and S. aureus) found in blood cultures within 6 hours after signalling growth. METHODS: The tube coagulase test was optimized and several latex agglutination tests were compared and evaluated before a validation period of 11 months was performed on consecutive positive blood culture patient samples from Kalmar County Hospital, Sweden. RESULTS: During the validation period 150 (91%) of a total of 166 gram-positive cocci (119 in clusters, 45 in chains or pairs and 2 undefined morphology) were correctly identified as S. aureus, CoNS, Pneumococci, Enterococci or group A streptococci (GAS), group B streptococci (GBS), group G streptococci (GGS) within 6 hours with a minimal increase in work-load and costs. The remaining samples (9%) were correctly identified during the next day. No samples were incorrectly grouped with this diagnostic strategy and no patient came to risk by early reporting. CONCLUSION: A simple strategy gives reliable and cost-effective reporting of >90% of the most common gram-positive cocci within 6 hours after a blood cultures become positive. The high specificity of the tests used makes preliminary reports reliable. The reports can be used to indicate the focus of infection and not the least, support faster administration of proper antimicrobial treatment for patients with serious bacterial infections.