Sören Gatermann

NDM-2 carbapenemase in Acinetobacter baumannii from Egypt

OBJECTIVES To analyse the mechanisms responsible for carbapenem resistance in one Acinetobacter baumannii isolate recovered from a patient transferred to Germany from an Egyptian hospital. METHODS PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of the isolate. RESULTS Sequencing of the PCR product obtained using primers for bla(NDM-1) revealed a variant of NDM-1 that had a C to G substitution at position 82 resulting in an amino acid substitution of proline to alanine at position 28. This variant was designated NDM-2. Genes encoding extended-spectrum β-lactamases or 16S RNA methylase were not detected. The strain lacked detectable plasmids and bla(NDM-2) was not transferred by conjugation. MLST showed that the isolate belonged to a new ST, ST103. CONCLUSIONS This work further underlines the spread of NDM carbapenemases in A. baumannii, and the spread of the corresponding gene in the Middle East. It also describes the first variant of NDM-1.

CLONING OF AAS, A GENE ENCODING A STAPHYLOCOCCUS SAPROPHYTICUS SURFACE PROTEIN WITH ADHESIVE AND AUTOLYTIC PROPERTIES

A gene encoding a novel cell wall‐associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N‐acetyl‐muramyl‐L‐alanine amidase and for an endo‐β‐N‐acetyl‐D‐glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N‐terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram‐positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.

Population Dynamics among Methicillin-Resistant Staphylococcus aureus Isolates in Germany during a 6-Year Period

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) originated from the health care setting but is now emerging in communities without health care contact (CA-MRSA) or in livestock (LA-MRSA). The impact on the whole MRSA population was assessed in a German prospective multicenter study. Thirty-three laboratories consecutively collected up to 50 MRSA isolates from infection or carriage during two sampling periods in 2004 to 2005 and 2010 to 2011. Patient-related data were collected using a standardized questionnaire. Methicillin resistance was confirmed by the detection of mecA or its homologue mecA LGA251. The spa type and major virulence factors were analyzed for each isolate. In total, 1,604 (2004 to 2005) and 1,603 (2010 to 2011) MRSA isolates were analyzed; one isolate from each sampling period harbored mecA LGA251. LA-MRSA increased significantly (odds ratio [OR] = 22.67, 95% confidence interval [CI] = 8.51 to 85.49, P < 0.0005) and spread over Germany, originating from northwestern regions. Panton-Valentine leukocidin-positive CA-MRSA rose significantly, particularly in southern Germany, but the proportion in 2010 to 2011 remained low (2.7%, OR = 2.80, 95% CI = 1.54 to 5.34, P < 0.0005). The emerging MRSA clones changed the MRSA population in Germany during a 6-year period significantly. The ongoing epidemiological shift and changes of MRSA sources create a need for revision of guidelines for MRSA infection control and treatment.

Identification of molecularly defined Staphylococcus aureus strains using matrix-assisted laser desorption/ionization time of flight mass spectrometry and the Biotyper 2.0 database

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced for bacterial identification. To our knowledge, this is the first study where the Biotyper 2.0 database (Bruker Daltonics) has been applied for bacterial identification in a local strain collection of molecularly defined Staphylococcus aureus. We showed that the accuracy of the Biotyper 2.0-based identification for 602 molecularly defined strains of S. aureus, irrespective of meticillin resistance, was equivalent to that of the molecularly defined reference even at a score cut-off value of 2. Also, 412 isolates of 20 different species of non-S. aureus staphylococci were all correctly identified to species level compared to the molecularly defined reference. Moreover, the MALDI-TOF MS-based S. aureus identification approach was clearly faster than more time-consuming methods such as a molecular identification approach.

Methicillin-resistant Staphylococcus aureus whole-body decolonization among hospitalized patients with variable site colonization by using mupirocin in combination with octenidine dihydrochloride

The object of this study was to investigate the efficacy of a methicillin-resistant Staphylococcus aureus (MRSA) multisite carriage decolonization in 32 hospitalized carriers--25 from surgical and seven from medical wards. Twenty-four of the patients had wounds (e.g. chronic ulcers, surgical sites) and 17 were spinal cord injury patients. Decolonization was performed by intranasal application of mupirocin, combined with an octenidine dihydrochloride bodywash over a period of five days. Samples from the nose, forehead, neck, axilla and groin were taken 24-48 h before beginning decolonization (sample point I, N=32) and 24-48 h afterwards (sample point II, N=32). Further samples, were taken seven to nine days after the procedure (sample point III, N=25). Contact sheep blood agar plates (24 cm2) were used to quantify MRSA colonies on forehead and neck. MRSA from other sample sites was determined semi-quantitatively. All patients were proven to be MRSA positive at one or more extranasal site(s); 18.8% did not have nasal carriage. The overall decolonization rate for all sites was 53.1% (sample point II) and 64% (sample point III), respectively. The reduction was significant for every site, showing a rate of 88.5% for nose (II, III) and of 56.3% (II) and 68% (III) for all extranasal sites together. Of 32 patients, a median of 6.5 cfu MRSA/24 cm2 was obtained for the forehead before decolonization and 0.5 cfu MRSA/24 cm2 for the neck. A significant reduction (0 cfu MRSA/24 cm2) from both sites was shown after treatment. Before decolonization procedures, median MRSA levels for the nose, groin and axilla were 55, 6 and 0 cfu/swab. After treatment, MRSA from each of these sites was significantly reduced. We conclude that nasal mupirocin combined with octenidine dihydrochloride whole-body wash is effective in eradicating MRSA from patients with variable site colonization.

Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR

ABSTRACT A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed.

Immunoblot analysis of immune response to Campylobacter pylori and its clinical associations.

Systemic immune response to Campylobacter pylori was detected by the immunoblot technique in serum samples from 200 patients, 129 blood donors, and 96 children. The results of the IgG immunoblot test showed excellent correlation with the detection of C pylori by culture and also with histopathological examination of the antrum, as well as with peptic ulcer disease. An IgA response also occurred and gave results comparable with those of the IgG immunoblot test, although on a quantitatively lower scale. The IgM immunoblots were of no help in the serodiagnosis of C pylori infection. The protein bands that seemed to be the most specific for C pylori and which were consistently observed in patients positive for C pylori were a 110 kilodalton and a 63 kilodalton band on the IgG immunoblot and an 89 kilodalton band on the IgA immunoblot. A 94 kilodalton and a 28 kilodalton band were also included in the evaluation. While immunoblot analysis may be used effectively for the serodiagnosis of C pylori infection and can distinguish between patients with normal antrum mucosa and those with gastritis, the test does not help to distinguish between those patients with antrum gastritis who subsequently develop peptic ulcers and those who do not.

Rapid Loss of Motility of Helicobacter pylori in the Gastric Lumen In Vivo

ABSTRACT The human pathogen Helicobacter pylori has infected more than half of the worlds population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization, H. pylori must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that Helicobacter spp. are known to survive low pH values for a certain time in vitro, the length of time that H. pylori persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found H. pylori to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of H. pylori in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of Helicobacter infection.

Animal shed Bacillus licheniformis spores possess allergy-protective as well as inflammatory properties

BACKGROUND Numerous epidemiologic studies have demonstrated an allergy-protective effect of farm life early in childhood. It has been hypothesized that environmental exposure to microbes may contribute to this effect. Because of their small size and thereby their potential for deposition in lower airways of small children, bacterial spores may be candidates for such allergy-protective effects. OBJECTIVE To investigate immune responses elicited by exposure to Bacillus spores in experimental settings. METHODS Animal shed and mattress dusts were analyzed for bacteria and fungi by aerobic and anaerobic growth. Bacillus licheniformis, the most prominent microorganism found in these samples, was investigated with respect to spore specific stimulation of pattern recognition receptors, monocyte-derived dendritic cells and T(H)-cell polarization in vitro as well as to the prevention of asthma development in a mouse model of allergic asthma. RESULTS In vitro, B. licheniformis spores activated a T(H)1 cytokine expression profile. In vivo application of these spores resulted in less spore-specific but long-lasting immune activation preventing eosinophilia and goblet cell hyperplasia; however, they provoked an influx of neutrophils in lung tissue of asthmatic mice. CONCLUSION Bacterial spores may contribute to the allergy-protective properties of farming environments, but their persistence in the lung causes ongoing immune activation in mouse experiments.

Comparison of phenotypic methods for penicillinase detection in Staphylococcus aureus

Penicillinase testing is required for Staphylococcus aureus isolates with a penicillin MIC of </=0.12 mg/L. This study compared five phenotypic assays with a PCR for blaZ when testing 197 S. aureus isolates. The starch-iodine plate method and nitrocefin tests had low sensitivities of 42.9% and 35.7%, respectively. The cloverleaf assay and the penicillin zone-edge determination method had sensitivities of 67.8% and 71.4%, respectively, and these methods might be appropriate in many circumstances, but were not as sensitive as blaZ PCR.