Employment of pqqE gene as molecular marker for the traceability of Gram negative phosphate solubilizing bacteria associated to plants.

Insoluble phosphorous compounds solubilization by soil bacteria is of great relevance since it puts available the phosphorus to be used by plants. The production of organic acids is the main microbiological mechanism by which insoluble inorganic phosphorus compounds are solubilized. In Gram negative bacteria, gluconic acid is synthesized by the activity of the holoenzyme glucose dehydrogenase-pyrroloquinoline quinine named GDH-PQQ. The use of marker genes is a very useful tool to evaluate the persistence of the introduced bacteria and allow to follow-up the effect of biotic and abiotic factors on these beneficial microorganisms in the soil. In previous studies we detected the presence of the pqqE gene in a great percentage of both non-culturable and culturable native soil bacteria. The objective of this study was to analyze the phylogeny of the sequence of pqqE gene and its potential for the study of phosphate solubilizing bacteria from pure and mixed bacterial cultures and rhizospheric soil samples. For this, the presence of the pqqE gene in the genome of phosphate solubilizing bacteria that belong to several bacteria was determined by PCR. Also, this gene was analyzed from mixed bacterial cultures and rhizospheric soil associated to peanut plants inoculated or not with phosphate solubilizing bacteria. For this, degenerate primers designed from several bacterial genera and specific primers for the genus Pseudomonas spp., designed in this study, were used. DNA template used from simple or mixed bacterial cultures and from rhizospheric soil samples was obtained using two different DNA extraction techniques. Results indicated that pqqE gene amplification product was found in the genome of all Gram negative phosphate solubilizing bacteria analyzed. It was possible to detect this gene in the DNA obtained from mixed cultures where these bacteria grew in interaction with other microorganisms and in that obtained from rhizospheric soil samples inoculated or not with these bacteria. The phylogenetic analysis indicated that pqqE gene is a conserved gene within related genera. In conclusion, pqqE gene could be a potential marker for the study of phosphate solubilizing bacterial populations.

Microbiological characterization of neuropathic diabetic foot infection: a retrospective study at a Portuguese tertiary hospital.

Diabetic foot infection imposes a significant burden and is the major cause of nontraumatic limb amputation. Adequate patient management with effective antibiotic therapy is crucial.This retrospective cohort study aimed to characterize the microbiology and resistance patterns of moderate to severe neuropathic diabetic foot infection in patients hospitalized at a tertiary referral hospital between January 2020 and June 2023. Deep tissue specimens from ulcers were collected for culture.Sixty inpatients were included (62% male, mean age 59.1 ± 11.5 years). Osteomyelitis was present in 90% of the patients. Among 102 microorganisms (average of 1.91 ± 1.25 pathogens per patient), 60.8% were gram-positive bacteria, 31.4% were gram-negative, 3.92% were anaerobic bacteria, and 3.92% were fungi. Staphylococcus aureus (19%) and Enterococcus faecium (17%) were the most common. Pseudomonas aeruginosa (8%) and bacteria of the Enterobacterales family (24%) accounted for all the isolated gram-negative bacteria. Sixteen percent of Staphylococcus aureus and 67% of coagulase-negative Staphylococci were resistant to methicillin. Resistance to ampicillin was found in 11% of Enterococci. All Pseudomonas aeruginosa isolates were sensitive to piperacillin-tazobactam, ceftazidime, or cefepime. Among the Enterobacterales, resistance rates were 35% for piperacillin-tazobactam, 38% for ceftazidime, 21% for cefepime, and 13% for carbapenems.Although the prevalence of methicillin-resistant staphylococci was lower than that in other studies, carbapenem resistance among gram-negative bacteria warrants attention. This study highlights the importance of understanding local epidemiology for effective diabetic foot infection management and resistance mitigation.

Microbiological analysis of tunneled hemodialysis catheters isolated from patients receiving hemodialysis in Saskatchewan.

Aim: To compare the microbial communities inside hemodialysis catheters from symptomatic and asymptomatic patients to determine their differences.Materials & methods: Catheters (n = 41) were removed from patients in the Saskatchewan Health Authority over an 18-month period. The catheter section inside the body was flushed and the contents were evaluated using culture-dependent and culture-independent analysis.Results: All catheters were colonized by bacteria, with considerable overlap between groups based on microbial communities and the individual species detected. More Gram-negative species were detected by sequencing, whereas predominantly Gram-positive strains were cultured. Antibiotic resistance and biofilm formation was widespread and not correlated with either catheter group.Conclusion: Common pathogens were detected in each set of catheters, therefore predicting infections based on the microbiology is difficult.

Many patients use catheters to help clean their blood, a process called hemodialysis. The use of catheters is also associated with complications, such as blood infections. We looked at the types of bacteria associated with catheters from patients who had infections (n = 21) and compared them to catheters from patients who had no signs of infection (n = 20). Once removed from the patient, we flushed out each catheter and tried to grow bacteria in different conditions. We also looked at DNA from within the catheter to identify bacterial species that were present. All 41 catheters had bacteria and there were many common species detected. We detected species known to cause illness such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas, Enterobacter, Morganella and Stenotrophomonas species. S. aureus was only grown from patients that had infections. Resistance to antibiotics was found to be common in bacteria grown from catheters. This did not seem to be influenced by whether patients were infected or not. Finally, we identified several catheters where two species, S. epidermidis and P. aeruginosa, were detected together. Our main conclusion was that bacteria are commonly present inside catheters that are used for hemodialysis, regardless of whether patients are infected or not.

Potentially pathogenic culturable bacteria in hemodialysis waters.

BACKGROUND: Hemodialysis patients are at risk of acquiring healthcare-related infections due to using non-sterile water to prepare hemodialysis fluid. Therefore, microbiological control and monitoring of used water are of crucial importance. MATERIALS AND METHODS: In this work, we identified bacterial populations occupying a hemodialysis water distribution system for almost a 6-month period in Ahvaz city, southwest of Iran. A total of 18 samples from three points were collected. We found high colony counts of bacteria on R2A agar. 31 bacteria with different morphological and biochemical characteristics were identified by molecular-genetic methods based on 16 S rRNA gene sequencing. Endotoxin concentrations were measured, using Endosafe® Rapid LAL Single-Test Vials. RESULTS: A diverse bacterial community was identified, containing predominantly Gram-negative bacilli. The most frequently isolated genus was Sphingomonas. Five species including M. fortuitum, M. lentiflavum, M.szulgai, M. barrassiae, and M. gordonae was identified .Despite the presence of Gram-negative bacteria the endotoxin analysis of all samples revealed that their endotoxin values were below the detection limit. CONCLUSION: The members of Sphingomonas genus along with Bosea and mycobacteria could be regarded as pioneers in surface colonization and biofilm creation. These bacteria with others like Pelomonas, Bradyrhizobium, staphylococcus, and Microbacterium may represent a potential health risk to patients under hemodialysis treatment.

Analysis of antibiotic resistance in Gram-negative bacilli in wild and exotic healthy birds in Brazil: A warning sign.

Bacterial antibiotic resistance is a public health problem affecting humans and animals. This study focuses on identifying Gram-negative bacilli (GNB) (MALDI-TOF MS and Klebsiella MALDI TypeR) resistant to antimicrobials in freshly emitted feces of healthy captive and rescued wild birds from a zoo in Brazil. Birds from the zoo and rescued from sixteen different orders were investigated. Resistant bacteria from feces were selected (MacConkey agar with 2 µg/mL cefotaxime). Genomic similarity and plasmid were investigated by Pulsed-Field Gel Electrophoresis of XbaI fragments (XbaI-PFGE) and S1-PFGE. Polymerase Chain Reaction (PCR) was performed to search for beta-lactamase genes. From 80 birds included, 26 from the zoo (50 %) and 18 rescued wild birds (64 %) presented cefotaxime-resistant GNB. E. coli and Klebsiella spp were the most prevalent species. Among 65 isolates from the zoo and rescued wild birds, 75 % were considered multidrug-resistant (MDR). The majority of the isolates were extended-spectrum beta-lactamases (ESBL) producing and resistant to enrofloxacin. blaCTX-M-GROUP-1, blaTEM, and blaSHV were the most detected genes, and blaKPC was detected in K. pneumoniae complex. According to genomic similarity results, some identical profiles were found in birds with no known contact among the zoo or rescued birds. Several isolates carried one to three plasmids (15-350 kb). The presence of multidrug-resistant (MDR) isolates from healthy captive and wild birds brings novel data on the dissemination of these elements to the environment.

Carbapenemase producing Gram negative bacteria: Review of resistance and detection methods.

Gram negative bacilli that are carbapenem resistant have emerged and are spreading worldwide. Infections caused by carbapenem resistant isolates posses a significant threat due to their high morbidity and mortality rates. Carbapenemases production by multi-drug resistant pathogens severely restricts treatment choices for illnesses caused by bacteria that are resistant to both carbapenems and majority of ß-lactam antibiotics. Various phenotypic and genotypic methods for identification can distinguish between different classes of carbapenemase and identify pathogens that are resistant to carbapenems. The establishment of a quick, accurate and reliable test for identifying the clinical strains that produce the carbapenemase enzyme is essential for optimum diagnosis of microbial pathogens and management of the global rise in the prevalence of carbapenemase producing bacterial strains. The aim of this review was to summarize the mechanisms of carbapenem resistance and to provide an overview of different carbapenemase detection methods for carbapenem resistant Gram negative bacilli.

Improved Blue Carba test and Carba NP test for detection and classification of major Class A and B carbapenemases, including dual producers, among Gram-negative bacilli.

Prompt and precise identification of carbapenemase-producing organisms is crucial for guiding clinical antibiotic treatments and limiting transmission. Here, we propose modifying the Blue Carba test (BCT) and Carba NP-direct (CNPd) to identify molecular carbapenemase classes, including dual carbapenemase strains, by adding specific Class A and Class B inhibitors. We tested 171 carbapenemase-producing Gram-negative bacilli strains-21 in Class A (KPC, NMC, SME), 58 in Class B (IMP, VIM, NDM, SPM), and 92 with dual carbapenemase production (KPC+NDM, KPC+IMP, KPC+VIM), all previously positive with BCT or CNPd. We also included 13 carbapenemase non-producers. ß-lactamases were previously characterized by PCR. The improved BCT/CNPd methods detect imipenem hydrolysis from an imipenem-cilastatin solution, using pH indicators and Class A (avibactam) and/or Class B (EDTA) inhibitors. Results were interpreted visually based on color changes. CNPd achieved 99.4% sensitivity and 100% specificity in categorizing carbapenemases, while BCT had 91.8% sensitivity and 100% specificity. Performance varied by carbapenemase classes: both tests classified all Class A-producing strains. For Class B, the CNP test identified 57/58 strains (98.3%), whereas the BCT test, 45/58 strains (77.6%), with non-fermenters posing the greatest detection challenge. For Classes A plus B dual producers, both tests performed exceptionally well, with only one indeterminate strain for the BCT. The statistical comparison showed both methods had similar times to a positive result, with differences based on the carbapenemase class or bacterial group involved. This improved assay rapidly distinguishes major Class A or Class B carbapenemase producers among Gram-negative bacilli, including dual-class combinations, in less than 2 hours. IMPORTANCE: Rapid and accurate identification of carbapenemase-producing organisms is of vital importance in guiding appropriate clinical antibiotic treatments and curbing their transmission. The emergence of negative bacilli carrying multiple carbapenemase combinations during and after the severe acute respiratory syndrome coronavirus 2 pandemic has posed a challenge to the conventional biochemical tests typically used to determine the specific carbapenemase type in the isolated strains. Several initiatives have aimed to enhance colorimetric methods, enabling them to independently identify the presence of Class A or Class B carbapenemases. Notably, no previous efforts have been made to distinguish both classes simultaneously. Additionally, these modifications have struggled to differentiate between carriers of multiple carbapenemases, a common occurrence in many Latin American countries. In this study, we introduced specific Class A and Class B carbapenemase inhibitors into the Blue Carba test (BCT) and Carba NP-direct (CNP) colorimetric assays to identify the type of carbapenemase, even in cases of multiple carbapenemase producers within these classes. These updated assays demonstrated exceptional sensitivity and specificity (≥ 90%) all within a rapid turnaround time of under 2 hours, typically completed in just 45 minutes. These in-house enhancements to the BCT and CNP assays present a rapid, straightforward, and cost-effective approach to determining the primary carbapenemase classes. They could serve as a viable alternative to molecular biology or immuno-chromatography techniques, acting as an initial diagnostic step in the process.

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Detection of KPC directly from positive blood cultures by MALDI-TOF: From research to the clinical microbiology laboratory routine.

Bloodstream infections (BSI) caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) are a subject of major clinical concern, mainly those associated with carbapenemase-producing isolates. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed to detect specific ß-lactamases, including KPC. We aimed to detect KPC enzyme directly from positive blood cultures using MALDI-TOF MS. Overall, 146 clinical Gram-negative bacilli (46 CR-GNB) recovered from consecutive blood cultures were evaluated. Proteins were extracted using formic acid, isopropyl alcohol, and water and spotted onto a steel target plate using the double-layer sinapinic acid method. The relative ions intensity ≥120 arbitrary units (a.u.) of a peak close to 28,700 m/z indicated the presence of KPC. The results were compared to HRM-qPCR methodology. This specific peak was observed in 11/14 blood bottles with blaKPC positive isolates (78.6% sensitivity), with 3 false-positive results (97.7% specificity). Analysis from colonies reached identical sensitivity (78.6%), but higher specificity (100%). The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid. It's excellent specificity indicates that positive results are consistently associated with the presence of a KPC producer in positive blood culture.

Gram-negative bacteremia, the risk factors, and outcome in children.

We aimed to determine the epidemiology and resistance patterns of Gram-negative bacteria, the risk factors and outcome of bloodstream infection (BSI). In all, 412 episodes in children who were hospitalized with the diagnosis of bacteremia were analyzed. The most common microorganisms were Klebsiella spp. (43.9%), Escherichia coli (13.5 %) and Acinetobacter spp. (10.6 %). Among isolates, 41.2 % were multidrug-resistant, 13.5 % were extensively drug-resistant and 0.4 % were pan-drug-resistant. Carbapenem resistance was revealed in 27.6 % of isolates. Carbapenem and colistin resistance increased over the years. The most common risk factors were the presence of a central-venous catheter and pediatric intensive care unit admission. Clinical response and infection-related mortality were significantly different in cases infected with carbapenem-resistant gram-negative (CRGN) vs carbapenem-susceptible gram-negative bacteria. The increase in multi-resistant Klebsiella spp. seems to be the biggest obstacles in fight against nosocomial infections. The increasing number of CRGN infections over the years affects both the clinical response and mortality rate of BSI.

Evaluation of an automated rapid phenotypic antimicrobial susceptibility testing (ASTar, Q-linea AB) applied directly on blood cultures bottles positive for Gram-negative pathogens.

We evaluated the performance of a new rapid phenotypic antimicrobial susceptibility test (ASTar; Q-linea AB) on Gram-negative bacilli, directly from positive blood cultures bottles. MIC values obtained by the routine reference method (Microscan, Beckman Coulter) were compared to the ones provided by the tested method (ASTar). ASTar demonstrated an overall essential agreement of 98% and a category agreement of 96.1%. The overall rate of major errors and very major errors was 2.5% and 3.3%, respectively. ASTar can represent a rapid, simple, and reliable method to speed up information about antimicrobial susceptibility of Gram-negative pathogens from positive blood culture bottles.

Prevalence of polymyxin-resistant bacterial strains in India: a systematic review and meta-analysis.

INTRODUCTION: Polymyxins, the cationic lipopeptide antibiotics, are the last line of therapeutics against the MDR Gram-negative bacterial (GNB) pathogens. Unfortunately, the rising cases of polymyxin-resistant strains from across the globe have adversely impacted their utility. While the molecular mechanisms responsible for developing polymyxin resistance (PolR) are largely understood, the prevalence of PolR strains in India has not been investigated systematically. The current study was undertaken to primarily determine the prevalence of PolR strains in India. Moreover, the extent of the spread of mobile colistin resistance (mcr) genes among the GNB strains in India was also determined. METHOD: A systematic search for articles using the relevant inclusion and exclusion criteria was performed in the applicable databases for the period January 2015 to December 2023. The included 41 studies were subjected to a meta-analysis using the Comprehensive Meta-Analysis software (V4.0). Publication biases were assessed using funnel plots and Egger's regression analysis. RESULT: Considering a total of 41 studies including 24 589 bacterial isolates the present meta-analysis found the rate of PolR bacteria in India to be at 15.0% (95% CI: 11.2 to 19.8). Among the Indian States, Tamil Nadu topped with the highest prevalence of PolR at 28.3%. Investigating the contribution of the mcr genes, it was observed that among the PolR strains, 8.4% (95% CI: 4.8 to 14.3) were mcr positive. CONCLUSION: The study determined the prevalence of PolR strains in India at 15.0%, which is higher than that of the global average at 10%. The study also determined that 8.4% of the PolR strains carried the mcr genes. The mcr-positive strains reported from India could be an underestimation of the actual numbers due to the non-inclusion of mcr screening in many previous studies. This study provides insight into the state of the PolR situation in India, which may be useful to develop a monitoring strategy to contain the spread of such strains and preserve the efficacy of the polymyxins.

Antimicrobial susceptibility patterns of urinary tract infections causing bacterial isolates and associated risk factors among HIV patients in Tigray, Northern Ethiopia.

BACKGROUND: Urinary tract infections, a prevalent global infectious disease, are clinical issues not well studied in HIV-positive individuals. UTIs have become a global drug resistance issue, but the prevalence and antibiotic susceptibility patterns of UTI-causing bacteria among HIV patients in Tigray, Ethiopia, are poorly understood. This study aims to identify the prevalence of UTI-causing bacteria, their antibiotic susceptibility patterns, and associated risk factors in HIV patients attending ART clinics at Mekelle General Hospital and Ayder Comprehensive Specialized Hospital in Tigray, Northern Ethiopia. METHOD: Clean-catch midstream urine samples (10-15 mL) were collected from HIV patients who are attending ART clinics at Mekelle General Hospital and Ayder Comprehensive Specialized Hospital. Samples were analyzed based on standard microbiological protocols using cysteine-lactose electrolyte deficient (CLED) agar. Pure colonies of bacterial isolates were obtained by sub-culturing into Mac-Conkey, Manitol Salt agar and blood agar plates. The bacterial isolates were then identified using macroscopic, microscopic, biochemical, and Gram staining methods. Gram-negative bacteria were identified using biochemical tests like triple sugar iron agar, Simon's citrate agar, lysine iron agar, urea, motility test, and indol test, whereas Gram-positive isolates were identified using catalase and coagulase tests. The Kirby-Bauer disk diffusion technique was used to analyze the antimicrobial susceptibility pattern of bacterial isolates. Data was analyzed using SPSS version 25.0. RESULTS: Among the 224 patients, 28 (12.5%) of them had been infected by UTIs-causing bacteria. E. coli was the dominant bacterium (16 (57%)) followed by K. pneumoniae (4 (14%)), and S. aureus (3 (11%)). Of the total bacterial isolates, 22 (78.6%) of them developed multi-drug resistance. All Gram-positive (100%) and 75% of Gram-negative bacterial isolates were found to be resistant to two or more drugs. Patients with a history of UTIs, and with CD4 count < 200 cells/ mm3, were more likely to have significant bacteriuria. Compared to male patients, female patients were more affected by the UTIs-causing bacteria. More than 93% of the UTIs-causing bacterial isolates were susceptible to nitrofurantoin, ceftriaxone, ciprofloxacin, and gentamycin; whereas they are highly resistant to ampicillin (96%), cotrimoxazole (82%) and tetracycline (71%). CONCLUSIONS: Most of the bacterial isolates were highly resistant to ampicillin, cotrimoxazole, and tetracycline. Female patients were more affected by the UTIs causing bacteria. The highest prevalence (12.5%) of UTIs in HIV patients needs special attention for better management and monitoring. Previous UTI history and immune suppression are predictors of UTIs, highlighting the need for intervention measures involving molecular studies to identify resistant bacteria genes and promote patient immune reconstitution.

Global trends in carbapenem- and difficult-to-treat-resistance among World Health Organization priority bacterial pathogens: ATLAS surveillance program 2018-2022.

OBJECTIVES: To report trends in carbapenem resistance and difficult-to-treat resistance (DTR) among clinical isolates of Gram-negative priority pathogens collected by the ATLAS global surveillance program from 2018 to 2022. METHODS: Reference broth microdilution testing was performed in a central laboratory for 79,214 Enterobacterales, 30,504 Pseudomonas aeruginosa, and 13,500 Acinetobacter baumannii-calcoaceticus complex isolates collected by a constant set of 157 medical centres in 49 countries in Asia Pacific (APAC), Europe (EUR), Latin America (LATAM), Middle East-Africa (MEA), and North America (NA) regions. MICs were interpreted by 2023 CLSI M100 breakpoints. ß-lactamase genes were identified for meropenem-nonsusceptible (MIC ≥2 mg/L) Enterobacterales isolates. RESULTS: Carbapenem-resistant Enterobacterales (CRE) detection increased (P < 0.05) in APAC, EUR, LATAM, and MEA regions and decreased in NA, while annual DTR percentages increased in all five regions. Carbapenem-resistant P. aeruginosa (CRPA; decreased in MEA region) and carbapenem-resistant A. baumannii-calcoaceticus complex (CRAB; decreased in MEA region and increased in EUR) remained relatively stable over time in all regions, although notably, annual percentages of CRAB and DTR A. baumannii-calcoaceticus complex isolates were consistently >25 percentage points lower in NA than in other regions. For all regions except NA, the majority of changes in CRE percentages could be attributed to hospital-acquired infections. Among meropenem-nonsusceptible Enterobacterales, KPC was the most frequent carbapenemase in NA and EUR each year. NDM was the most prevalent carbapenemase detected in 2022 in other global regions. CONCLUSION: CRE, CRPA, CRAB, and DTR rates vary among global regions over time highlighting the need for continuing surveillance to inform treatment strategies and antimicrobial stewardship.

Development and evaluation of test methods for the detection and enumeration of opportunistic waterborne pathogens from the hospital environment.

BACKGROUND: Many Gram-negative bacteria other than Pseudomonas aeruginosa have been implicated in waterborne outbreaks, but standardized laboratory detection methods for these organisms have not been established. AIM: This study aimed to establish laboratory testing methodologies for six waterborne pathogens: Acinetobacter spp., Burkholderia spp., Cupriavidus spp., Delftia acidovorans, Elizabethkingia spp. and Stenotrophomonas maltophilia. METHODS: Water samples were spiked by UK Health Security Agency laboratories and sent to the Glasgow Royal Infirmary laboratory for analysis. Water samples were spiked with either a pure culture of target organism or the target organism in water containing normal background flora, to ensure that the methodology could identify organisms from a mixed culture. Volumes of 100 mL were filtered under negative pressure on to culture media and incubated at 30 °C and 37 °C. The incubation time was 7 days, with plates read on days 2, 5 and 7. Further identification of colonies was undertaken using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). FINDINGS: Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar, chocolate bacitracin agar and pseudomonas selective agar. The optimal temperature for isolation was 30 °C. The optimal incubation time was 5 days, and MALDI-TOF MS identified all test species reliably. CONCLUSION: The methodology described was able to detect the six tested waterborne pathogens reliably, and can be utilized by laboratories involved in testing water samples during outbreak investigations.

Molecular characterization and descriptive analysis of carbapenemase-producing Gram-negative rod infections in Bogota, Colombia.

In this study, the genetic differences and clinical impact of the carbapenemase-encoding genes among the community and healthcare-acquired infections were assessed. This retrospective, multicenter cohort study was conducted in Colombia and included patients infected with carbapenem-resistant Gram-negative rods between 2017 and 2021. Carbapenem resistance was identified by Vitek, and carbapenemase-encoding genes were identified by whole-genome sequencing (WGS) to classify the alleles and sequence types (STs). Descriptive statistics were used to determine the association of any pathogen or gene with clinical outcomes. A total of 248 patients were included, of which only 0.8% (2/248) had community-acquired infections. Regarding the identified bacteria, the most prevalent pathogens were Pseudomonas aeruginosa and Klebsiella pneumoniae. In the WGS analysis, 228 isolates passed all the quality criteria and were analyzed. The principal carbapenemase-encoding gene was blaKPC, specifically blaKPC-2 [38.6% (88/228)] and blaKPC-3 [36.4% (83/228)]. These were frequently detected in co-concurrence with blaVIM-2 and blaNDM-1 in healthcare-acquired infections. Notably, the only identified allele among community-acquired infections was blaKPC-3 [50.0% (1/2)]. In reference to the STs, 78 were identified, of which Pseudomonas aeruginosa ST111 was mainly related to blaKPC-3. Klebsiella pneumoniae ST512, ST258, ST14, and ST1082 were exclusively associated with blaKPC-3. Finally, no particular carbapenemase-encoding gene was associated with worse clinical outcomes. The most identified genes in carbapenemase-producing Gram-negative rods were blaKPC-2 and blaKPC-3, both related to gene co-occurrence and diverse STs in the healthcare environment. Patients had several systemic complications and poor clinical outcomes that were not associated with a particular gene.IMPORTANCEAntimicrobial resistance is a pandemic and a worldwide public health problem, especially carbapenem resistance in low- and middle-income countries. Limited data regarding the molecular characteristics and clinical outcomes of patients infected with these bacteria are available. Thus, our study described the carbapenemase-encoding genes among community- and healthcare-acquired infections. Notably, the co-occurrence of carbapenemase-encoding genes was frequently identified. We also found 78 distinct sequence types, of which two were novel Pseudomonas aeruginosa, which could represent challenges in treating these infections. Our study shows that in low and middle-income countries, such as Colombia, the burden of carbapenem resistance in Gram-negative rods is a concern for public health, and regardless of the allele, these infections are associated with poor clinical outcomes. Thus, studies assessing local epidemiology, prevention strategies (including trials), and underpinning genetic mechanisms are urgently needed, especially in low and middle-income countries.

A decade of neonatal sepsis in Stockholm, Sweden: Gram-positive pathogens were four times as common as Gram-negatives.

PURPOSE: To assess Gram-positive bacterial (GPB) bloodstream infection (BSI) in neonates, covering incidence, morbidity, mortality, antimicrobial resistance patterns and biomarkers in Region Stockholm, Sweden between 2006 and 2016. METHODS: A population-based retrospective epidemiological study including infants with GPB-BSI, admitted to the neonatal units at Karolinska University Hospital (KUH). Data were collected from patient records, the Swedish Neonatal Quality Register, the microbiological laboratory at KUH and the Swedish Public Health Agency. RESULTS: We identified 357 infants with GPB-BSI, representing an incidence of 1.47/1000 live births (LB). Group B streptococcus (GBS) was the most common pathogen causing BSI in full-term infants and early-onset sepsis (EOS) (0.20/1000 LB), while coagulase-negative staphylococci (CoNS) were predominant in infants born very preterm and in late-onset sepsis (LOS) (0.79/1000 LB). There were no fatal GBS BSI cases, but 10.2% developed meningitis. The GPB case fatality rate was 9.5% and the sepsis fatality rate 2.8%. In GPB-BSI, 1/10 did not have an elevated C-reactive protein level. Staphylococcus aureus (S. aureus) BSI increased during the study period, but no methicillin or vancomycin resistant strains were found. The antimicrobial resistance (AMR) rate was highest in CoNS isolates. CONCLUSION: GPB-BSI was four times more common than Gram-negative BSI in neonates but resulted in lower mortality rate. GBS was the most common pathogen in full-term infants and in EOS. CoNS was the most common pathogen in LOS and infants born very preterm, and the AMR rate was high in these isolates. The increasing trend of S. aureus BSI indicates a need of further investigation.

Persistent Gram-negative Bloodstream Infection Increases the Risk of Recurrent Bloodstream Infection With the Same Species.

The association between persistent gram-negative bloodstream infection (GN-BSI), or ongoing positive cultures, and recurrent GN-BSI has not been investigated. Among 992 adults, persistent GN-BSI was associated with increased recurrent GN-BSI with the same bacterial species and strain (6% vs 2%; P = .04). Persistent GN-BSI may be a marker of complicated infection.