Leclercia pneumoniae sp. nov., a bacterium isolated from clinical specimen in Leipzig, Germany.

Strain 49125T was isolated from an infant with pneumonia and septicaemia at the Leipzig University Hospital. Phenotypic and genomic traits were investigated. The strain's biochemical profile and its MALDI-TOF spectrogram did not differ from comparative samples of Leclercia adecarboxylata, thus far the sole member of the Leclercia species. A circular genome with a size of 4.4 Mbp and a G+C content of 55.0 mol% was reconstructed using hybrid Illumina and Nanopore sequencing. Phylogenetic analysis was based on 172 marker genes and validated using a k-mer-based search against a large genome collection including subsequent in silico DNA-DNA hybridization. Whole genome average nucleotide identity to any described species was below 95%, suggesting that strain 49125T represents a new species, for which we propose the name Leclercia pneumoniae sp. nov. with the type strain 49125T (=LMG 32245T=DSM 112336T).

Bacteremia after professional mechanical plaque removal in patients with chronic periodontitis.

OBJECTIVE: The aim of this study was to investigate the characteristics of bacteremia caused by professional mechanical plaque removal (PMPR) in two groups of patients with generalized moderate chronic periodontitis. MATERIALS AND METHODS: Venous blood samples were taken at multiple time points for one hour following PMPR in fifty patients with generalized moderate chronic periodontitis. Subjects consisted of two groups, one group was receiving supportive periodontal therapy (SPT, n = 25) and the other group was receiving initial periodontal therapy (IPT, n = 25). Blood samples were processed and analyzed for cultivable microflora. Pertinent clinical parameters were recorded for each patient in both groups. RESULTS: Bacteremia was detected in 10 of 25 SPT and 8 of 25 IPT patients (p = 0.796). In both groups, the prevalence of bacteremia was dependent on the time of blood sampling and varied in magnitude between <102  CFU/ml and 106  CFU/ml. Sixteen different bacterial species were identified in both groups, mostly Actinomyces naeslundii (SPT n = 3, IPT n = 4) and Streptococcus spp. (SPT n = 6, IPT n = 2). In regression models, Grade II furcation involvement (p = 0.004) and Gingival Bleeding Index (p = 0.036) had affected the occurrence of bacteremia but in the SPT group only. CONCLUSION: Professional mechanical plaque removal was associated with bacteremia regardless of whether a patient was receiving SPT or IPT.

Discrimination of Human Pathogen Clostridium Species Especially of the Heterogeneous C. sporogenes and C. botulinum by MALDI-TOF Mass Spectrometry.

Clostridium species cause several local and systemic diseases. Conventional identification of these microorganisms is in part laborious, not always reliable, time consuming or does not always distinguish different species, i.e., C. botulinum and C. sporogenes. All in, there is a high interest to find out a reliable, powerful and rapid method to identify Clostridium spp. not only on genus but also on species level. The aim of the present study was to identify Clostridium spp. strains and also to find differences and metabolic groups of C. botulinum by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). A total of 123 strains of Clostridium spp. (C. botulinum, n = 40, C. difficile, n = 11, C. tetani, n = 11, C. sordellii, n = 20, C. sporogenes, n = 18, C. innocuum, n = 10, C. perfringens, n = 13) were analyzed by MALDI-TOF MS in combination with methods of multivariate statistical analysis. MALDI-TOF MS analysis in combination with methods of multivariate statistical analysis was able to discriminate between the different tested Clostridium spp., even between species which are closely related and difficult to differentiate by traditional methods, i.e., C. sporogenes and C. botulinum. Furthermore, the method was able to separate the different metabolic groups of C. botulinum. Especially, E gene-positive C. botulinum strains are clearly distinguishable from the other species but also from those producing other toxin types. Thus, MALDI-TOF MS represents a reliable and above all quick method for identification of cultivated Clostridium species.

Identification of Clostridium species using the VITEK® MS.

The genus Clostridium is of high clinical relevance, as some species may cause rapid and even lethal infections. Thus, a timely identification of these anaerobic bacteria is desirable. Conventional identification methods rely on biochemical properties of these organisms, however, establishing these is time-consuming and not always reliable. Alternatively, 16S rRNA gene sequence based diagnostic methods may be used, but they are expensive and not ubiquitously available. This study was designed to assess the possibility to identify Clostridium species employing the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, 848 Clostridium strains representing 42 species were analyzed with the VITEK® MS instrument (bioMérieux, Marcy l'Etoile, France), comparing mass spectra derived from these organisms with the spectra provided in the available database. 90.3% of the strains were correctly identified at species level and another 3.6% at genus level. Since the number of Clostridium species included in the database was rather limited (21 altogether), the spectra obtained were also analyzed employing the Shimadzu Pro Series software. Thus, it became possible to create a dendrogram of the species included in this study.

In vitro activity of tigecycline and comparators against a European collection of anaerobes collected as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) 2010-2016.

The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) is a global program that aims to monitor the in vitro antimicrobial activities of current therapeutic agents against clinical isolates. This study presents surveillance data for Gram-positive and Gram-negative anaerobic isolates (N = 7008) collected from nine European countries between 2010 and 2016. Presented in this study are antimicrobial susceptibility data, according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST) breakpoints, and minimum inhibitory concentration (MIC) distributions. The antimicrobial agents tested were cefoxitin (Gram-negative isolates only), clindamycin, meropenem, metronidazole, penicillin (Gram-positive isolates only), piperacillin-tazobactam and tigecycline. Among all Gram-positive and Gram-negative anaerobes, the lowest rates of resistance were to meropenem and metronidazole (0.0%-1.7% and 0.0%-1.9%, respectively). High rates of resistance were reported to clindamycin, in particular among isolates of the Bacteroides fragilis group (22.1%-48.1%) and Prevotella spp. (10.9%-32.2%). The majority of MIC distributions were unimodal, with the exception of clindamycin, which were mostly bimodal. Fifty percent of Gram-negative isolates gave tigecycline MICs between 0.06 and 1 mg/L, and 50% of Gram-positive isolates exhibited tigecycline MICs between 0.06 and 0.25 mg/L. The findings of this study suggest that the majority of anaerobic isolates were susceptible to meropenem and metronidazole, and that tigecycline remained active, but clindamycin resistance is a cause for concern in Europe. Surveillance studies, such as T.E.S.T., provide information on changes in the susceptibility of clinically important pathogens to commonly prescribed antimicrobial agents, and can highlight problems of antimicrobial resistance that need to be addressed.

[Decolonization of Health Care Workers In a Neonatal Intensive Care Unit Carrying a Methicillin-Susceptible Staphylococcus aureus Isolate]. / Dekolonisation bei Nachweis von Methicillin-sensiblen Staphylococcus aureus bei Mitarbeitern einer neonatologischen Intensivstation.

BACKGROUND: In 2016, an increased rate of methicillin-susceptible Staphylococcus aureus colonization was detected on a neonatal intensive care unit at the Leipzig University Hospital. Typing results showed a predominant spa-type t091. Considering nosocomial clustering, several infection prevention measures (e. g. intensified standard precautions, single-occupancy room, cohorted patients, continuing education of staff) were introduced, including staff screening followed by decolonization of colonized health care workers. METHODS: Staff members showing positive on screening carried out a 5-day decolonization program at home. Decolonization products containing octenidine as active ingredient were used first. At the earliest, 48 h after completing the procedure, the success of the intervention was tested (3 buccal and nasal swabs were taken on consecutive days). If 2 attempts at decolonization were not successful, staff members were provided with a mupirocin-containing nasal ointment instead of octenidine. RESULTS: Of 128 employees examined, 43 (33.6%) were identified as carriers of S. aureus. In 9 cases (20.9%; 9/43) the S. aureus matched with type t091. 2 carriers (4.7%; 2/43) of MRSA were detected as well. The first decolonization attempt against t091 and MRSA failed altogether. After a second decolonization, 3 cases became negative. Finally, 8 remaining staff members were decolonized successfully with mupirocin containing nasal ointment. CONCLUSIONS: Various reasons might explain the difficulties of decolonization such as the challenge of managing decolonization at home, inhibitory factors as well as inconsistent performance of decolonization measures. Additionally, differences between the preparations for the nasal decontamination may be considered.

Environmental pollution with antimicrobial agents from bulk drug manufacturing industries in Hyderabad, South India, is associated with dissemination of extended-spectrum beta-lactamase and carbapenemase-producing pathogens.

PURPOSE: High antibiotic and antifungal concentrations in wastewater from anti-infective drug production may exert selection pressure for multidrug-resistant (MDR) pathogens. We investigated the environmental presence of active pharmaceutical ingredients and their association with MDR Gram-negative bacteria in Hyderabad, South India, a major production area for the global bulk drug market. METHODS: From Nov 19 to 28, 2016, water samples were collected from the direct environment of bulk drug manufacturing facilities, the vicinity of two sewage treatment plants, the Musi River, and habitats in Hyderabad and nearby villages. Samples were analyzed for 25 anti-infective pharmaceuticals with liquid chromatography-tandem mass spectrometry and for MDR Gram-negative bacteria using chromogenic culture media. In addition, specimens were screened with PCR for bla VIM, bla KPC, bla NDM, bla IMP-1, and bla OXA-48 resistance genes. RESULTS: All environmental specimens from 28 different sampling sites were contaminated with antimicrobials. High concentrations of moxifloxacin, voriconazole, and fluconazole (up to 694.1, 2500, and 236,950 µg/L, respectively) as well as increased concentrations of eight other antibiotics were found in sewers in the Patancheru-Bollaram industrial area. Corresponding microbiological analyses revealed an extensive presence of extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae and non-fermenters (carrying mainly bla OXA-48, bla NDM, and bla KPC) in more than 95% of the samples. CONCLUSIONS: Insufficient wastewater management by bulk drug manufacturing facilities leads to unprecedented contamination of water resources with antimicrobial pharmaceuticals, which seems to be associated with the selection and dissemination of carbapenemase-producing pathogens. The development and global spread of antimicrobial resistance present a major challenge for pharmaceutical producers and regulatory agencies.

Antimicrobial Susceptibility among European Gram-Negative and Gram-Positive Isolates Collected as Part of the Tigecycline Evaluation and Surveillance Trial (2004-2014).

BACKGROUND: European centers (n = 226) involved in the Tigecycline Evaluation and Surveillance Trial (TEST, 2004-2014) submitted data and bacterial isolates. METHODS: Minimal inhibitory concentrations and susceptibility were determined using Clinical and Laboratory Standards Institute methods and European Committee on Antimicrobial Susceptibility Testing breakpoints. RESULTS: The rates of the following resistant pathogens increased from 2004 to 2014: extended-spectrum ß-lactamase (ESBL)-positive Escherichia coli (from 8.9 to 16.9%), multidrug-resistant Acinetobacter baumannii complex (from 15.4 to 48.5%), and ESBL-positive Klebsiella pneumoniae (from 17.2 to 23.7%). The rate of methicillin-resistant Staphylococcus aureus was 27.5% in 2004 and 28.9% in 2014. Resistance to the carbapenems (imipenem and meropenem) was 37.4 and 14.5% for A. baumannii complex and Pseudomonas aeruginosa, respectively. Carbapenem resistance was ≤4.3% among the Enterobacteriaceae and 0.2% against Streptococcus pneumoniae. The resistance to tigecycline ranged between 7.4% against ESBL-producing K. pneumoniae and 0.0% against S. aureus. CONCLUSIONS: The carbapenems and tigecycline were active against Enterobacteriaceae. Agents with activity against A. baumannii complex and P. aeruginosa are limited. The carbapenems, tigecycline, linezolid, and vancomycin were active against Gram-positive organisms.

Prevalence of Actinomyces spp. in patients with chronic periodontitis.

This study investigated the prevalence of Actinomyces spp. in shallow, deep and very deep pockets of patients with chronic periodontitis compared to healthy controls and correlated the results with clinical status. Twenty patients with chronic periodontitis and 15 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. From each patient samples of supra and subgingival plaque were taken separately from teeth with shallow, deep and very deep pockets. Samples of supragingival plaque and sulcular microflora were collected from the healthy subjects. All the samples were cultivated on different media at 37̊C in an anaerobic atmosphere for 7 days. All the suspect colonies were identified using a rapid ID 32 A system (bioMèrieux) and MALDI-TOF-MS analysis using an Autoflex II Instrument (Bruker Daltonics) together with in house developed identification software and a reference spectra database. A total of 977 strains were identified as Actinomyces. Actinomyces naeslundii/oris/johnsonii (430 isolates) was the most prevalent species and was found in all patients and in almost all of the healthy subjects. Significant differences (p=0.003) between the groups were found for Actinomyces odontolyticus/meyeri and Actinomyces israelii which were associated with periodontitis patients. Actinomyces dentalis was found in higher percentage (p=0.015) in the periodontitis group. Actinomyces gerencseriae and Actinomyces massiliensis were significantly more often found supragingivally than subgingivally (p=0.004, p=0.022, respectively) in the periodontitis group. Whether some Actinomyces species, definitely important plaque formers, are actively involved in the pathogenicity of chronic periodontitis needs further investigation.

Colonization with extended-spectrum beta-lactamase-producing and carbapenemase-producing Enterobacteriaceae in international travelers returning to Germany.

Two hundred and twenty-five healthy German volunteers traveling to 53 different countries (mostly in Asia, Africa and South America) were enrolled in a prospective cohort study. Stool samples and data on potential travel-associated risk factors (such as type of travel, nutritional habits, occurrence of gastroenteritis) were collected before and after traveling. Screening for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) and carbapenemase-producing Enterobacteriaceae (CPE) was performed using selective media (CHROMagar™ ESBL/CPE plates). Isolates with confirmed ESBL-phenotype were examined for the presence of blaCTX-M, blaTEM, blaSHV, and blaVIM, blaIMP, blaNDM, blaKPC, blaOXA-48 genes by PCR amplification and sequencing. Antimicrobial susceptibility testing was performed using conventional microbroth dilution. Pre-travel analysis of 205 fully evaluable participants revealed an ESBL-PE prevalence rate of 6.8% (14/205). Among 191 participants that were ESBL-negative before travel, 58 (30.4%) were colonized by ESBL-producing Escherichia coli, and 5 (8.6%) additionally carried ESBL-producing Klebsiella pneumoniae upon return. However, no carbapenem-resistant Enterobacteriaceae were detected. ESBL-genotyping revealed that 52/54 (96.6%) E. coli and 4/4 (100%) K. pneumoniae strains available for sequencing produced CTX-M enzymes, mostly CTX-M-15 (33/56, 58.9%), and 2/54 (3.7%) E. coli strains produced SHV-12 enzymes. Travel to India was associated with the highest ESBL-PE acquisition rate (11/15, 73.3%; p=0.015), followed by South East Asia (22/46, 47.8%; p=0.038). Evaluation of travel-associated risk factors demonstrated significance for the occurrence of gastroenteritis (p=0.011). Strictly practiced hand hygiene and exclusive consumption of packaged beverages showed no protective effect. The ESBL-PE persistence rate after 6 months was 8.6% (3/35). We conclude that global efforts are needed to address the further spread of ESBL-PE in the community. Active surveillance and contact isolation precautions may be recommended at admission to medical facilities especially for patients who traveled to India and South East Asia in the previous 6 months.

Drug resistance of Mycobacterium tuberculosis isolates from tuberculosis lymphadenitis patients in Ethiopia.

BACKGROUND & OBJECTIVES: The emergence of drug resistance tuberculosis (TB) is a significant challenge for TB control and prevention programmes, and the major problem is multidrug resistant tuberculosis (MDR-TB). The present study was carried out to determine the frequency of drug resistant Mycobacterium tuberculosis isolates among newly and retreated TB lymphadenitis patients and risk factors for acquiring this infection. METHODS: Two hundred twenty five M. tuberculosis isolates from TB lymphadenitis patients who were diagnosed as new and retreated tuberculosis cases between April 2012 and May 2012 were included in this study. Isolates were tested for susceptibility to isoniazed (INH), rifampicin (RMP), streptomycin (SM), ethambutol (EMB) and pyrazinamide (PZA) using the BacT/AlerT 3D system protocol. RESULTS: Among 225 isolates, 15 (6.7%) were resistant to at least one first line anti-TB drug. Three (1.3%) were MDR-TB. Resistance to INH, RMP, SM, and EMB was found in 8 (3.6%), 4 (1.8%), 10 (4.4%), and 4 (1.8%) isolates, respectively. Of the 212 new TB lymphadenitis cases three (1.4%) were MDR-TB. A rifampicin resistant M. tuberculosis isolate was diagnosed from smear and culture negative newly treated cases. All isolates were susceptible to PZA. Matted cervical lymph nodes were the prominent sites involved. Newly treated TB lymphadenitis patients had a greater risk for presenting resistance to anti-TB drugs ( p =0.046). INTERPRETATION & CONCLUSIONS: Our study showed that TB lymphadenitis patients harboured drug resistant TB and MDR-TB, although at a low rate. Resistance was not associated with age, sex, patients' education and contact history. Further research is required to determine transmission dynamics of drug resistant strains.

Early-Outcome Differences between Acute and Chronic Periprosthetic Joint Infections-A Retrospective Single-Center Study.

Periprosthetic joint infections (PJI) are serious complications after arthroplasty, associated with high morbidity, mortality, and complex treatment processes. The outcomes of different PJI entities are largely unknown. The aim of this study was to access the early outcomes of different PJI entities. A retrospective, single-center study was conducted. The characteristics and outcomes of patients with PJI treated between 2018 and 2019 were evaluated 12 months after the completion of treatment. Primary endpoints were mortality, relapse free survival (RFS) and postoperative complications (kidney failure, sepsis, admission to ICU). A total of 115 cases were included [19.1% early (EI), 33.0% acute late (ALI), and 47.8% chronic infections (CI)]. Patients with ALI were older (p = 0.023), had higher ASA scores (p = 0.031), preoperative CRP concentrations (p = 0.011), incidence of kidney failure (p = 0.002) and sepsis (p = 0.026). They also tended towards higher in-house mortality (ALI 21.1%, 13.6% EI, 5.5% CI) and admission to ICU (ALI 50.0%, 22.7% EI, 30.9% CI). At 12 months, 15.4% of patients with EI had a relapse, compared to 38.1% in ALI and 36.4% in CI. There are differences in patient characteristics and early outcomes between PJI entities. Patients with EI have better early clinical outcomes. Patients with ALI require special attention during follow-up because they have higher occurrences of relapses and postoperative complications than patients with EI and CI.

Molecular epidemiology and transmission dynamics of Mycobacterium tuberculosis in Northwest Ethiopia: new phylogenetic lineages found in Northwest Ethiopia.

BACKGROUND: Although Ethiopia ranks seventh among the world's 22 high-burden tuberculosis (TB) countries, little is known about strain diversity and transmission. In this study, we present the first in-depth analysis of the population structure and transmission dynamics of Mycobacterium tuberculosis strains from Northwest Ethiopia. METHODS: In the present study, 244 M. tuberculosis isolates where analysed by mycobacterial interspersed repetitive unit - variable number tandem repeat 24-loci typing and spoligotyping methods to determine phylogenetic lineages and perform cluster analysis. Clusters of strains with identical genotyping patterns were considered as an indicator for the recent transmission. RESULTS: Of 244 isolates, 59.0% were classified into nine previously described lineages: Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%). Interestingly, 31.6% of the strains were grouped into four new lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating a high rate of recent transmission. CONCLUSIONS: This study confirms a highly diverse M. tuberculosis population structure, the presence of new phylogenetic lineages and a predominance of the Dehli/CAS lineage in Northwest Ethiopia. The high rate of recent transmission indicates defects of the TB control program in Northwest Ethiopia. This emphasizes the importance of strengthening laboratory diagnosis of TB, intensified case finding and treatment of TB patients to interrupt the chain of transmission.

Magnitude of gene mutations conferring drug resistance in mycobacterium tuberculosis isolates from lymph node aspirates in ethiopia.

OBJECTIVE: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients. METHODS: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType(®) MTBDRplus and GenoType® MTBDRsl assay. RESULTS: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay. CONCLUSION: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.

Extended-spectrum beta-lactamases found in Escherichia coli isolates obtained from blood cultures and corresponding stool specimen.

With extended-spectrum ß-lactamases (ESBLs) and CTX-M enzymes being on the rise, antimicrobial treatment of enterobacterial infections is becoming more and more challenging. Our study aimed at a molecular characterization of phenotypically ESBL-positive E. coli strains obtained from blood cultures of patients of the University Hospital of Leipzig (UKL), Germany. The presence of CMY-2, CTX-M-14 and CTX-M-15 was investigated using Streck ARM-D Kit (Streck, USA). Real-time amplifications were performed by QIAGEN Rotor-Gene Q MDx Thermocycler (QIAGEN, Thermo Fisher Scientific, USA). Antibiograms as well as epidemiological data were evaluated. Among 117 cases, 74.4% of the isolates showed a resistance to ciprofloxacin, piperacillin and ceftazidime or cefotaxime while being susceptible to imipenem/meropenem. The proportion of ciprofloxacin resistance was significantly higher than the proportion of ciprofloxacin susceptibility. At least one of the investigated genes was detected in 93.1% of the blood culture E. coli isolates: CTX-M-15 (66.7%), CTX-M-14 (25.6%) or the plasmid-mediated ampC gene CMY-2 (3.4%). 2.6% were tested positive for two resistance genes. 94 of the corresponding stool specimens tested positive for ESBL producing E. coli (94/112, 83.9%). 79 (79/94, 84%) E. coli strains found in the stool samples matched with the respective patient's blood culture isolate phenotypically (MALDI-TOF, antibiogram). The distribution of resistance genes was in accordance with recent studies in Germany as well as worldwide. This study provides indications of an endogenous focus of infection and emphasize the importance of screening programs for high-risk patients.

In vitro activities of clindamycin, imipenem, metronidazole, and piperacillin-tazobactam against susceptible and resistant isolates of Bacteroides fragilis evaluated by kill kinetics.

The aim of the present study was to investigate the activities of clindamycin, imipenem, metronidazole, and piperacillin-tazobactam against 12 Bacteroides fragilis isolates (resistant and susceptible strains) by kill kinetics over 24 h. In contrast to the other antimicrobial agents, clindamycin did not affect strains with MICs of >8.0 µg/ml. For those strains with MICs of ≤ 8.0 µg/ml, all employed antibiotics except clindamycin showed nearly bactericidal activity. Metronidazole proved to be the most active antimicrobial agent.

Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia.

BACKGROUND: The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system. METHODS: Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany. RESULTS: Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance. CONCLUSION: The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. However, the GenoType® MTBDRsl assay shows limitations in detecting resistance to EMB.

A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry.

BACKGROUND: Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. MATERIAL/METHODS: We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. RESULTS: Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. CONCLUSIONS: This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics.

A real-time PCR for the detection and characterisation of Aspergillus species.

An early diagnosis of an invasive fungal infection is essential for the initiation of a specific antifungal therapy and to avoid unnecessary discontinuation of a baseline therapy for haematological or oncological diseases. A real-time PCR assay for the detection and strain identification of Aspergillus species from culture strains was evaluated. DNA preparation was evaluated in contaminated culture media, urine and serum. A LightCycler PCR to differentiate various Aspergillus species was established. A real-time PCR assay for the detection of Aspergillus species was improved and was able to detect and differentiate medically important Aspergillus spp. The sensitivity of the test was <10 plasmid equivalents/assay. The real-time PCR assay is a useful tool for the rapid identification of Aspergillus species and might be useful as an early diagnostic tool to detect an invasive fungal infection. A real-time PCR protocol was improved by generating plasmid standards, additional generation of melting curves for species identification and the correlation between the melting temperature and the nucleotide exchanges within the used 18S rRNA gene region.

Chronic Clostridium botulinum infections in farmers.

Although botulism is usually an acute, often lethal disease that is caused by the ingestion of botulinum neurotoxin, there are also recognized forms like infant botulism, wound botulism, or "botulism of undefined origin" that are characterized by the fact that Clostridium botulinum colonizes the host and produces its toxin in the host. Evidence is presented here that a disease in cattle and in human care takers of diseased animals that has evolved over the past two decades, may be a chronic, visceral form of C. botulinum infection.