Phenotypic and genotypic characteristics of beta-lactamase dominant with CARBA, AmpC, and ESBL-producing bacteria in municipal wastewater influent in Helsinki, Finland.

OBJECTIVES: Analysing samples of municipal wastewater influent (before treatment) can help to map the status of antibiotic-resistant bacteria (ARB) at the population level in sewershed communities and may also help in predicting the public health risks of ARB in surface water because of the outfall of wastewater. In this study, we investigated the bacterial isolates carrying beta-lactamase genes in wastewater and compared their genotypic and phenotypic characteristics. METHODS: A total of 399 bacterial isolates grown on CHROMagarESBL (n = 207) and CHROMagarKPC (n = 192) from composite wastewater influent samples (n = 7) from the Viikinmäki wastewater treatment plant (Helsinki) were subcultured, nucleic acid was extracted, and the prevalence of different beta-lactamase genes was screened with multiplex polymerase chain reaction (PCR). All PCR-positive isolates were identified with MALDI-TOF. RESULTS: A total of 32.6% of isolates (130 of 399) were PCR positive for at least one resistance gene, and 13% of these positive isolates out of 130 had at least three resistance genes. Among the 22 detected genes, blaGES group was the most prevalent, at 25.8% (n = 198; many isolates carried multiple genes), followed by blaMOX (13.1%) and blaTEM (10.1%) as most frequently detected. Furthermore, out of 18 different bacterial species/genera detected as carrying beta-lactamase genes, A. hydrophila/caviae (28.5%), Enterobacter spp. (16.9%), and E. coli (14.6%) were the most prevalent. Enterobacter spp., Aeromonas spp., and K. cryocescens potentially carried AmpC genes, and E. coli carried ESBL genes. CONCLUSION: We recorded a huge variety of beta-lactamases (blaAmpC, blaESBL, and blaCARBA) genes in many potential pathogens that probably originated from both enteric and environmental sources.

Phenotypic Detection of Carbapenemase and AmpC-ß-Lactamase Production among Extended Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli and Klebsiella spp. Isolated from Clinical Specimens.

Introduction: Data on antimicrobial resistance (AMR) are sparse across numerous African countries, as microbiological analyses are not routinely conducted and surveillance data are not collected. Accordingly, clinical samples are not routinely tested for carbapenem-resistant bacteria and, therefore, the general understanding of their prevalence in the region remains limited. Methods: Between January 2020 and June 2022, we collected extended spectrum ß-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) isolates from five hospitals in Burkina Faso. After an initial culture on ESBL-selective media, the species were identified using API20E and isolates were tested against 13 antimicrobial agents using the disc diffusion method on Mueller-Hinton (MH) agar. ESBL production was confirmed via a double-disc synergy test. Production of carbapenemases and AmpC-ß-lactamases and phenotypic co-resistance were determined. Results: Among the 473 ESBL-PE, 356 were ESBL-E. coli (ESBL-Ec) and 117 were Klebsiella spp. (ESBL-K). Of these isolates, 5.3% were carbapenemase and 5.3% were AmpC-ß-lactamase-positive. Three types of carbapenemases were identified: 19 NDM, 3 OXA-48-like and 1 VIM. Two isolates produced both NDM and OXA-48-like carbapenemases. Carbapenemase producers were detected at all levels of healthcare. Co-resistance rates were up to 85% for aminoglycosides, 90% for sulfonamides, 95% for fluoroquinolones and 25% for chloramphenicol. Fosfomycin resistance was 6% for ESBL-Ec and 49% for ESBL-K (49%). Conclusions: Some of the ESBL-Ec and ESBL-K co-produced carbapenemases and/or AmpC-ß-lactamases at all healthcare levels and in various sample types with high co-resistance rates to non-betalactams. Carbapenem resistance is no longer rare, calling for testing in routine diagnostics, a comprehensive resistance surveillance system and infection control within healthcare.

Wastewater Surveillance Detected Carbapenemase Enzymes in Clinically Relevant Gram-Negative Bacteria in Helsinki, Finland; 2011-2012.

Antimicrobial resistance profiling of pathogens helps to identify the emergence of rare or new resistance threats and prioritize possible actions to be taken against them. The analysis of wastewater (WW) can reveal the circulation of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARG) among the catchment communities. Here, we analyzed WW influent samples to determine the prevalence of carbapenemase genes-carrying Gram-negative bacteria (Carba-GNB) in Helsinki, Finland. This study set important historical reference points from the very early stage of the carbapenemase era, during the period 2011-2012. A total of 405 bacterial isolates grown on CHROMagarKPC (n = 195) and CHROMagarESBL (n = 210) from WW influent samples were collected between October 2011 and August 2012 and were analyzed. The bacterial DNA from the isolates was extracted, and the prevalence of carbapenemases genes bla KPC, bla NDM, bla GES, bla OXA-48, bla IMP, bla IMI, and bla VIM were screened with multiplexed PCR. All carbapenemase-positive isolates were identified taxonomically to species or genus level with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The nucleic acid extraction was successful for 399 isolates, of which 59 (14.8%) were found to carry carbapenemase genes. A total of 89.8% of the carbapenemase positive isolates (53 out of 59) were obtained from CHROMagarKPC plates and only 10.2% (six out of 59) were obtained from CHROMagar ESBL plates. Among the Carba-GNB isolates, 86.4% were bla GES (51 out of 59), 10.2% were bla KPC (six out of 59), and 3.4% were bla VIM (two out of 59). The most common carba-gene, bla GES, was carried by 10 different bacterial species, including Aeromonas spp., Enterobacter spp., and Kluyvera spp.; the bla KPC gene was carried by Escherichia coli, Klebsiella pneumoniae, and Kluyvera cryocescens; and the bla VIM gene was carried by Aeromonas hydrophila/caviae and Citrobacter amalonaticus. This study emphasizes that wastewater surveillance (WWS) can be an additional tool for monitoring antimicrobial resistance (AMR) at the population level.

Prevalence of diarrhoeal pathogens among children under five years of age with and without diarrhoea in Guinea-Bissau.

BACKGROUND: Childhood diarrhoea, a major cause of morbidity and mortality in low-income regions, remains scarcely studied in many countries, such as Guinea-Bissau. Stool sample drying enables later qPCR analyses of pathogens without concern about electricity shortages. METHODS: Dried stool samples of children under five years treated at the Bandim Health Centre in Bissau, Guinea-Bissau were screened by qPCR for nine enteric bacteria, five viruses, and four parasites. The findings of children having and not having diarrhoea were compared in age groups 0-11 and 12-59 months. RESULTS: Of the 429 children- 228 with and 201 without diarrhoea- 96.9% and 93.5% had bacterial, 62.7% and 44.3% viral, and 52.6% and 48.3% parasitic pathogen findings, respectively. Enteroaggregarive Escherichia coli (EAEC; 60.5% versus 66.7%), enteropathogenic E. coli (EPEC; 61.4% versus 62.7%), Campylobacter (53.2% versus 51.8%), and enterotoxigenic E. coli (ETEC; 54.4% versus 44.3%) were the most common bacterial pathogens. Diarrhoea was associated with enteroinvasive E. coli (EIEC)/Shigella (63.3%), ETEC (54.4%), astrovirus (75.0%), norovirus GII (72.6%) and Cryptosporidium (71.2%). The only pathogen associated with severe diarrhoea was EIEC/Shigella (p<0.001). EAEC was found more frequent among the infants, and EIEC/Shigella, Giardia duodenalis and Dientamoeba fragilis among the older children. CONCLUSIONS: Stool pathogens proved common among all the children regardless of them having diarrhoea or not.

Bacterial, viral and parasitic pathogens analysed by qPCR: Findings from a prospective study of travellers' diarrhoea.

BACKGROUND: The diagnostics of travellers' diarrhoea (TD) has been revolutionised by multiplex qPCR assays. While mostly of bacterial aetiology, viruses and parasites account for the disease among 10-20% of travellers. Despite this, prospective studies applying qPCR assays remain scarce that cover not only bacteria, such as the various diarrhoeagenic Escherichia coli (DEC), but also viral and parasitic pathogens. METHOD: We analysed by qPCR pre- and post-travel stool samples of 146 Finnish travellers for bacterial, viral and parasitic pathogens: enteropathogenic (EPEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC), and enteroinvasive (EIEC) E. coli; Shigella, Campylobacter, Salmonella, Yersinia and Vibrio cholerae; norovirus G1 and G2, rotavirus, enteroviruses, and sapovirus; and Giardia lamblia, Entamoeba histolytica, and Cryptosporidium. Symptoms and medication data during travel were collected by questionnaires. RESULTS: We detected bacterial pathogens in 102/146 samples (69.9%; EAEC, EPEC, ETEC most common), viral ones in 13 (8.9%; norovirus most common), and parasitic ones in one (0.7%; Giardia). Noroviruses were associated with severe symptoms (23.5% versus non-severe 4.9%). In the TD group, 41.7% (5/12) of those with viral pathogens (vs. 13.3%; 11/83 without) took antibiotics. CONCLUSION: Viral pathogens, particularly noroviruses, prevail in severe TD. The symptoms of viral disease are often severe and lead to unwarranted use of antibiotics.

Clinical aspects of heat-labile and heat-stable toxin-producing enterotoxigenic Escherichia coli: A prospective study among Finnish travellers.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing travellers' diarrhoea (TD) among visitors to low- and middle-income countries (LMIC). Scant data are available on rates of travel-acquired ETEC producing heat-labile (LT) and/or heat-stable (ST) toxin or its subtypes, STh (human) and STp (porcine) in various geographic regions, and on clinical pictures associated with each toxin. METHODS: Using qPCR, we analysed LT, STh, and STp in stools positive for ETEC in a prospective study among 103 Finnish travellers visiting LMIC. They filled in questionnaires and provided stool samples before and after travel. We scrutinized geographic distribution of LT, STh, and STp ETEC findings, and association between these different ETEC subtypes and moderate/severe TD. RESULTS: Among the 103 stool samples positive for ETEC toxins, the rate for LT was 76%, for STh 26%, and STp 41%. The rate for LT-only was 44%, for STh-only 6%, STp-only 16%, LT + STh 10%, LT + STp 15%, STh + STp 3%, and LT + STh + STp 8%. Findings varied by destination; the rates of LT, STh, and STp were 79%, 21%, and 57%, respectively, in Southern Asia (n = 14); 85%, 10%, and 20% in South-eastern Asia (n = 20); 84%, 13%, and 29% in Eastern Africa (n = 31); and 56%, 50%, and 63% in Western Africa (n = 32), respectively. Of travellers positive for LT, STh, and STp, 83%, 100%, and 88%, encountered TD; 35%, 55%, and 41% reported moderate/severe TD. STh was associated with moderate/severe TD. CONCLUSIONS: Toxin findings varied by destination; multiple toxins were commonly detected. Moderate/severe TD was reported most frequently by subjects with STh-ETEC.

Reactive arthritis and other musculoskeletal symptoms associated with acquisition of diarrhoeagenic Escherichia coli (DEC).

OBJECTIVES: Using a prospective research design, we evaluated the association between acquisition of diarrhoeagenic Escherichia coli (DEC) and development of reactive arthritis (ReA) and other reactive musculoskeletal (MSK) symptoms among international travellers. METHODS: A total of 526 study participants were asked to provide pretravel and post-travel stool samples and fill in questionnaires (pretravel, post-travel and 3-week follow-up). A multiplex quantitative PCR assay was deployed to detect five DEC comprising enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, enterohaemorrhagic E. coli and enteroinvasive E. coli and Salmonella, Shigella, Campylobacter, Yersinia, and Vibrio cholerae. Multivariate analysis was employed to identify factors predisposing to MSK symptoms. New post-travel MSK symptoms reported by participants with DEC were assessed by phone interviews and, if needed, clinically confirmed. RESULTS: From among the total of 224 volunteers who returned all questionnaires and stool specimens, 38 (17.0%) reported MSK symptoms. Multivariate analysis revealed that acquisition of DEC was associated with MSK symptoms (OR 3.9; 95% CI 1.2 to 13.3). Of the 151 with only-DEC, four (2.6%) had ReA, two (1.3%) reactive tendinitis and three (2.0%) reactive arthralgia. ReA was mostly mild, and all patients with ReA were negative for human leucocyte antigen B27. Antibiotic treatment of travellers' diarrhoea did not prevent development of MSK symptoms. CONCLUSION: A total of 17% of volunteers reported post-travel MSK symptoms. DEC acquisition was associated with an increased risk of developing them, yet the ReA incidence remained low and the clinical picture mild. Antibiotic treatment did not protect against development of MSK symptoms.

Despite Predominance of Uropathogenic/Extraintestinal Pathotypes Among Travel-acquired Extended-spectrum ß-Lactamase-producing Escherichia coli, the Most Commonly Associated Clinical Manifestation Is Travelers' Diarrhea.

BACKGROUND: One-third of the 100 million travelers to the tropics annually acquire extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE), with undefined clinical consequences. METHODS: Symptoms suggesting Enterobacteriaceae infections were recorded prospectively among 430 Finnish travelers, 90 (21%) of whom acquired ESBL-PE abroad. ESBL-PE isolates underwent polymerase chain reaction-based detection of diarrheagenic Escherichia coli (DEC) pathotypes (enteroaggregative E. coli [EAEC], enteropathogenic E. coli [EPEC], enterotoxigenic E. coli [ETEC], enteroinvasive E. coli, and Shiga toxin-producing E. coli), and extraintestinal pathogenic/uropathogenic E. coli (ExPEC/UPEC). Laboratory-confirmed ESBL-PE infections were surveyed 5 years before and after travel. RESULTS: Among the 90 ESBL-PE carriers, manifestations of Enterobacteriaceae infection included travelers' diarrhea (TD) (75/90 subjects) and urinary tract infection (UTI) (3/90). The carriers had 96 ESBL-producing E. coli isolates, 51% exhibiting a molecular pathotype: 13 (14%) were DEC (10 EAEC, 2 EPEC, 1 ETEC) (12 associated with TD) and 39 (41%) ExPEC/UPEC (none associated with UTI). Of ESBL-PE, 3 (3%) were ExPEC/UPEC-EAEC hybrids (2 associated with diarrhea, none with UTI). Potential ESBL-PE infections were detected in 15 of 90 subjects (17%). The 10-year medical record survey identified 4 laboratory-confirmed ESBL-PE infections among the 430 travelers, all in subjects who screened ESBL-PE negative after returning home from their index journeys but had traveled abroad before their infection episodes. CONCLUSIONS: Half of all travel-acquired ESBL-producing E. coli strains qualified molecularly as pathogens. Extraintestinal and uropathogenic pathotypes outnumbered enteric pathotypes (41% vs 14%), yet the latter correlated more closely with symptomatic infection (0% vs 92%). Despite more ESBL-PE strains qualifying as ExPEC/UPEC than DEC, travel-acquired ESBL-PE are more often associated with TD than UTI.

Despite antibiotic treatment of travellers' diarrhoea, pathogens are found in stools from half of travellers at return.

BACKGROUND: Among visitors to the (sub)tropics, 20-50% contract travellers' diarrhoea (TD) and 5-30% take antibiotics. While shortening the duration of illness, antimicrobials predispose to acquisition of multi-drug resistant bacteria. Therefore, liberal use is no longer advocated. Although antibiotics kill pathogens, no data support the view that they could prevent post-infectious sequelae. We investigated how antibiotic use for TD abroad impacts the pathogen findings at return. MATERIALS AND METHODS: We revisited 456 travellers' clinical data and stool pathogens examined by qPCR for Salmonella, Yersinia, Campylobacter, Shigella, Vibrio cholerae and enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC) and enteroinvasive (EIEC) Escherichia coli. RESULTS: Among travellers with TD, antibiotic users had pathogen-positive samples less frequently than non-users (50% versus 83%). The difference was significant for EPEC (23% versus 47%) and EAEC (27% versus 54%), but not ETEC (17% versus 26%) or the other pathogens. Shigella/EIEC was found more often among antibiotic users than non-users (4% versus 1%). CONCLUSION: Despite antibiotic treatment of TD, half of the users still had stool pathogens at return, reflecting either antibiotic resistance of pathogens or recolonisation/reinfection while abroad. Treatment of TD with antibiotics during travel should not be interpreted to indicate eradication of pathogens.

Travellers' diarrhoea: Impact of TD definition and control group design on study results.

BACKGROUND: Travellers' diarrhoea (TD) is a common health problem among visitors to the (sub)tropics. Much research deals with aetiology, prevention, and post-infection sequalae, yet the data may not allow comparisons due to incompatible definitions of TD and No TD control groups. METHOD: The impact of defining TD and No TD control groups was explored by revisiting our recent data. We set up two TD groups: classical TD i.e. ≥3 loose or liquid stools/day and WHO TD (diarrhoea as defined by the WHO) i.e. any diarrhoea, and four No TD groups by TD definition and timing (no classical/WHO TD during travel, no ongoing classical/WHO TD). RESULTS: TD was recorded for 37% versus 65% of subjects when using classical versus WHO definitions, respectively; the proportions of the various pathogens proved similar. The strictest criterion for the No TD control group (no WHO TD during travel) yielded pathogens among 61% and the least strict (no ongoing classical TD) among 73% of the travellers; the differences were greatest for enteroaggregative Escherichia coli and Campylobacter. CONCLUSIONS: Definition of TD and control group design substantially impact on TD study results. The WHO definition yields more cases, but the pathogen selection is similar by both definitions. Design of the No TD control group was found critical: only those remaining asymptomatic throughout the journey should be included.

Potential Diarrheal Pathogens Common Also in Healthy Children in Angola.

BACKGROUND: Globally, diarrhea kills almost 1500 children daily. In diagnostics, molecular methods are replacing traditional assays. We aimed to investigate enteropathogens in children with and without diarrhea in Luanda, the capital of Angola. METHODS: One hundred and ninety-four stool samples from 98 children with acute diarrhea and 96 children without diarrhea were investigated for 17 enteropathogens with multiplex real-time polymerase chain reaction. RESULTS: The median age of children was 10.5 months. Enteropathogens, bacteria, viruses and parasites were detected in 91%, 78%, 50% and 25%, respectively. A positive finding was significantly (P = 0.003) more common in diarrhea when testing for all pathogens combined, for bacteria alone and for viruses alone. More than one pathogen was found more frequently in diarrhea than in non-diarrhea stool samples, in 87% and in 59% (P < 0.0001), respectively. The median number (interquartile range) of pathogens detected was 3 (2) versus 1.5 (2; P < 0.0001), respectively. When age was taken into account, diarrhea was found to be associated with enterotoxigenic and enteroaggregative Escherichia coli, Shigella, Campylobacter, rotavirus, sapovirus and Cryptosporidium. CONCLUSIONS: Multiplex polymerase chain reaction detected enteropathogens in almost all stool samples of children in Luanda, albeit this occurred more often in diarrhea. Children with diarrhea showed more mixed infections than children without diarrhea.

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea.

BACKGROUND: In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. METHODS: A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. RESULTS: The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. CONCLUSIONS: Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Fluoroquinolone antibiotic users select fluoroquinolone-resistant ESBL-producing Enterobacteriaceae (ESBL-PE) - Data of a prospective traveller study.

BACKGROUND: One third of travellers to the poor regions of the (sub)tropics become colonized by extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE). Co-resistance to non-beta-lactam antibiotics complicates the treatment of potential ESBL-PE infections. METHODS: We analysed co-resistance to non-beta-lactams among travel-acquired ESBL-PE isolates of 90 visitors to the (sub)tropics with respect to major risk factors of colonization: destination, age, travellers' diarrhoea (TD) and antibiotic (AB) use. RESULTS: Of the ESBL-PE isolates, 53%, 52%, 73%, and 2% proved co-resistant to ciprofloxacin, tobramycin, co-trimoxazole, and nitrofurantoin, respectively. The rates were similar among those with (TD+) or without (TD-) travellers' diarrhoea. Among fluoroquinolone-users vs. AB non-users, the co-resistance rates for ciprofloxacin were 95% versus 37% (p = 0.001), for tobramycin 85% versus 43% (p = 0.005), co-trimoxazole 85% versus 68% (p = 0.146), and nitrofurantoin 5% versus 2% (p = 0.147). In multivariable analysis co-resistance to ciprofloxacin was associated with increasing age, fluoroquinolone use, and tobramycin resistance. CONLUSIONS: While TD predisposes to ESBL-PE non-selectively, antimicrobial use favours strains resistant to drug taken and, simultaneously, any drug with resistance genetically linked to the drug used. Antibiotics taken during travel predispose to ESBL-PE with a high co-resistance rate.

Increased Risk for ESBL-Producing Bacteria from Co-administration of Loperamide and Antimicrobial Drugs for Travelers' Diarrhea.

Antimicrobial drug treatment of travelers' diarrhea is known to increase the risk for colonization with extended-spectrum ß-lactamase-producing Enterobacteriaceae. Among 288 travelers with travelers' diarrhea, the colonization rate without medications was 21%. For treatment with loperamide only, the rate was 20%; with antimicrobial drugs alone, 40%; and with loperamide and antimicrobial drugs, 71%.

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles.

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19-20 (FH19-20) and 5-7 (FH5-7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5-7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5-7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5-7. We found that although FH5-7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5-7 domains. Furthermore, binding of FH5-7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5-7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5-7 and regulates alternative pathway activation on plasma HDL particles.

Differentiating virulent 027 and non-027 Clostridium difficile strains by molecular methods.

OBJECTIVE: Hypervirulent Clostridium difficile clade has been shown to include several lineages of ribotype 027 and also other ribotypes. We present data on additional non-027 strains, identified as presumptive 027 by two commercial molecular C. difficile assays. METHODS: The tested clinical isolates were selected from the national reference laboratory collection on the basis of toxin gene profile similarities with ribotype 027 and tested with XpertC. difficile/Epi and Amplidiag C. difficile+027 assay. RESULT: Xpert misclassified five ribotypes (016, 019, 080, 176 and variant of type 046) as presumptive 027 and Amplidiag two ribotypes (016, 176). The misclassified strains were rare, covering 1.6% of reference laboratory strain collection. CONCLUSION: Our findings confirm the concept that there are closely related outliers to hypervirulent 027 clones that can be misclassified as 027, and that these comprise numerous ribotypes, including previously reported four ribotypes (198, 176, 244, 019), and additional three (016, v046, 080) identified in the present study.

Bacterial infections in patients with type 1 diabetes: a 14-year follow-up study.

OBJECTIVE: This study explored the annual occurrence/incidence of bacterial infections, and their association with chronic hyperglycemia and diabetic nephropathy, in patients with type 1 diabetes. DESIGN: In a register-based follow-up study, we investigated the frequency of bacterial infections in patients with type 1 diabetes (n=4748) and age-matched and sex-matched non-diabetic control (NDC) subjects (n=12 954) using nationwide register data on antibiotic drug prescription purchases and hospital discharge diagnoses, collected between 1996 and 2009. Diabetic nephropathy was classified based on the urinary albumin excretion rate (AER). RESULTS: The hospitalization rate due to bacterial infections was higher in patients with diabetes compared with NDCs (rate ratio (RR) 2.30 (95% CI 2.11 to 2.51)). The rate correlated with the severity of diabetic nephropathy: RR for microalbuminuria was 1.23 (0.94 to 1.60), 1.97 (1.49 to 2.61) for macroalbuminuria, 11.2 (8.1 to 15.5) for dialysis, and 6.72 (4.92 to 9.18) for kidney transplant as compared to patients with diabetes and normal AER. The annual number of antibiotic purchases was higher in patients with diabetes (1.00 (1.00 to 1.01)) as compared with NDCs (0.47 (0.46 to 0.47)), RR=1.71 (1.65 to 1.77). Annual antibiotic purchases were 1.18-fold more frequent in patients with microalbuminuria, 1.29-fold with macroalbuminuria, 2.43-fold with dialysis, and 2.74-fold with kidney transplant as compared to patients with normal AER. Each unit of increase in glycated hemoglobin was associated with a 6-10% increase in the number of annual antibiotic purchases. CONCLUSIONS: The incidence of bacterial infections was significantly higher in patients with type 1 diabetes compared with age-matched and sex-matched NDC subjects, and correlated with the severity of diabetic nephropathy in inpatient and outpatient settings.

Antimicrobials increase travelers' risk of colonization by extended-spectrum betalactamase-producing Enterobacteriaceae.

BACKGROUND: More than 300 million travelers visit regions with poor hygiene annually. A significant percentage of them become colonized by resistant intestinal bacteria such as extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) and may transmit the strains to others and to medical care settings when they return home. Despite the threats to global healthcare caused by an upsurge in antimicrobial resistance, no effort has been centered on prevention of colonization while traveling. METHODS: Stool samples were collected from 430 Finns before and after traveling outside Scandinavia. All specimens were analyzed for ESBL- and carbapenemase-producing Enterobacteriaceae (CPE). Questionnaires were used to survey volunteers about use of antimicrobials as well as other potential risk factors. The results were subjected to multivariable analysis. RESULTS: Twenty-one percent (90/430) of the travelers became colonized by ESBL-PE and none by CPE. Geographic region, occurrence of travelers' diarrhea (TD), age, and use of antimicrobial (AB) for TD were identified as independent risk factors predisposing to contracting ESBL-PE. Eleven percent of those in subgroup TD-AB-, 21% in TD+AB-, and 37% in TD+AB+ acquired ESBL-PE. The risk proved to be highest in South Asia (46%); 23% became colonized in subgroup TD-AB-, 47% in TD+AB-, and 80% in TD+AB+. In Southeast Asia, the rates were 14%, 37%, and 69%, respectively. CONCLUSIONS: TD and antimicrobials for TD proved to be independent risk factors, with up to 80% of TD+AB+ travelers contracting ESBL-PE. In modern pre-travel counseling for those visiting high-risk regions, travelers should be advised against taking antibiotics for mild or moderate TD.

High-fat meals induce systemic cytokine release without evidence of endotoxemia-mediated cytokine production from circulating monocytes or myeloid dendritic cells.

AIMS: Dietary fats have been shown to promote the translocation of bacterial endotoxins from the gut into circulation, which may induce systemic inflammation and modulate the inflammatory response of circulating immune cells. The aim of this study was to determine the effect of the postprandial milieu on inflammation and the inflammatory response of antigen presenting cells in the context of type 1 diabetes (T1D). MATERIALS AND METHODS: Eleven patients with T1D and eleven nondiabetic controls were recruited as part of the FinnDiane study and given two fatty meals during 1 day. Cytokine responses in monocytes and myeloid dendritic cells (mDCs) as well as serum lipopolysaccharide activity levels, triglyceride concentrations and cytokine concentrations were measured from fasting and postprandial blood samples. RESULTS: Postprandially, patients with T1D and controls showed significant increases in eight inflammatory cytokines (IL-6, TNF-α, IL-1ß, IFN-α, IL-10, IFN-γ, IL-12 and MIP-1ß) without concomitant increase in serum LPS activity. Serum cytokine production was similar in both groups. No postprandial change was seen in the IL-6, TNF-α or IL-1ß production of mDCs or monocytes. At fasting, diabetic mDCs exhibited higher LPS-induced IL-6 and IL-1ß production than controls. CONCLUSIONS: Acute high-fat meals increase circulating cytokines but have no effect on serum lipopolysaccharide activity levels or cytokine production in circulating mDCs or monocytes. Our results suggest that postprandial increase in serum cytokine levels is neither mediated by circulating endotoxins nor the activation of circulating innate cells. The production of high-fat meal-induced inflammatory markers is most likely regulated at the tissue level.

Patients with type 1 diabetes show signs of vascular dysfunction in response to multiple high-fat meals.

BACKGROUND: A high-fat diet promotes postprandial systemic inflammation and metabolic endotoxemia. We investigated the effects of three consecutive high-fat meals on endotoxemia, inflammation, vascular function, and postprandial lipid metabolism in patients with type 1 diabetes. METHODS: Non-diabetic controls (n = 34) and patients with type 1 diabetes (n = 37) were given three high-caloric, fat-containing meals during one day. Blood samples were drawn at fasting (8:00) and every two hours thereafter until 18:00. Applanation tonometry was used to assess changes in the augmentation index during the investigation day. RESULTS: Three consecutive high-fat meals had only a modest effect on serum LPS-activity levels and inflammatory markers throughout the day in both groups. Of note, patients with type 1 diabetes were unable to decrease the augmentation index in response to the high-fat meals. The most profound effects of the consecutive fat loads were seen in chylomicron and HDL-metabolism. The triglyceride-rich lipoprotein remnant marker, apoB-48, was elevated in patients compared to controls both at fasting (p = 0.014) and postprandially (p = 0.035). The activities of the HDL-associated enzymes PLTP (p < 0.001), and CETP (p = 0.007) were higher and paraoxonase (PON-1) activity, an anti-oxidative enzyme bound to HDL, decreased in patients with type 1 diabetes (p = 0.027). CONCLUSIONS: In response to high-fat meals, early signs of vascular dysfunction alongside accumulation of chylomicron remnants, higher augmentation index, and decreased PON-1 activity were observed in patients with type 1 diabetes. The high-fat meals had no significant impact on postprandial LPS-activity in non-diabetic subjects or patients with type 1 diabetes suggesting that metabolic endotoxemia may be more central in patients with chronic metabolic disturbances such as obesity, type 2 diabetes, or diabetic kidney disease.