IncA/C Plasmid Carrying bla(NDM-1), bla(CMY-16), and fosA3 in a Salmonella enterica Serovar Corvallis Strain Isolated from a Migratory Wild Bird in Germany.

A Salmonella enterica serovar Corvallis strain was isolated from a wild bird in Germany. This strain carried the IncA/C2 pRH-1238 plasmid. Complete sequencing of the plasmid was performed, identifying the blaNDM-1, blaCMY-16, fosA3, sul1, sul2, strA, strB, aac(6')-Ib, aadA5, aphA6, tetA(A), mphA, floR, dfrA7, and merA genes, which confer clinically relevant resistance to most of the antimicrobial classes, including ß-lactams with carbapenems, fosfomycin, aminoglycosides, co-trimoxazole, tetracyclines, and macrolides. The strain likely originated from the Asiatic region and was transferred to Germany through the Milvus migrans migratory route.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium.

AIMS: To analyse genetic changes in the oafA gene explaining the loss of O5-antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:-. METHODS AND RESULTS: The oafA gene in 52 O5-antigen-negative and 77 O5-antigen-positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:- (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5-antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5-antigen-negative strains could be identified. In 77% of the O5-antigen-negative strains, a 7-bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3'end of undetermined size was responsible for the lack of O5-antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. CONCLUSIONS: O5-antigen-negative Salm. Typhimurium and Salm. 4,[5],12:i:- strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5-antigen expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The loss of O5-antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5-antigen-positive and O5-antigen-negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i-) strains.

High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types.

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.

Use of multiple-locus variable-number tandem-repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections.

AIMS: To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple-locus variable-number tandem-repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients. METHODS AND RESULTS: A total of 102 isolates of Salm. Typhimurium phage type DT41 were MLVA typed. MLVA typing showed 4, 12, 25, 9 and 8 different alleles at the five MLVA loci 9, 5, 6, 10 and 3, respectively. A dendrogram based on MLVA types was constructed, and one large group, nine minor groups and 29 more unrelated MLVA types were obtained. The major group included 20 of the 30 human isolates. Isolates obtained from broiler breeders demonstrated major diversity, indicating the existence of several independent introductions of DT41 at farm level. When comparison was made to isolates included from Germany and England, DT41 seems to be ubiquitous in the wild fauna which might represent a risk factor for poultry. CONCLUSIONS: Transmission from Danish broilers to humans was not demonstrated, neither was the transmission from rearing farms to broiler breeder farms. Sources of infection at broiler breeder farm level remained unidentified. SIGNIFICANCE AND IMPACT OF THE STUDY: Major diversity was demonstrated for DT41 MLVA types. A persisting problem with infection of broiler breeder flocks with DT41 was not reflected in broiler flocks originating from these flocks.

[Tasks and duties of veterinary reference laboratories for food borne zoonoses]. / Aufgaben der veterinärmedizinischen Referenzlaboratorien im Bereich der lebensmittelbedingten Zoonosen.

Reference laboratories are of central importance for consumer protection. Field expertise and high scientific competence are basic requirements for the nomination of a national reference laboratory. To ensure a common approach in the analysis of zoonotic hazards, standards have been developed by the reference laboratories together with national official laboratories on the basis of Art. 33 of Directive (EG) No. 882/2004. Reference laboratories function as arbitrative boards in the case of ambivalent or debatable results. New methods for detection of zoonotic agents are developed and validated to provide tools for analysis, e. g., in legal cases, if results from different parties are disputed. Besides these tasks, national reference laboratories offer capacity building and advanced training courses and control the performance of ring trials to ensure consistency in the quality of analyses in official laboratories. All reference laboratories work according to the ISO standard 17025 which defines the grounds for strict laboratory quality rules and in cooperation with the respective Community Reference Laboratories (CRL). From the group of veterinary reference laboratories for food-borne zoonoses, the national reference laboratories are responsible for Listeria monocytogenes, for Campylobacter, for the surveillance and control of viral and bacterial contamination of bivalve molluscs, for E. coli, for the performance of analysis and tests on zoonoses (Salmonella), and from the group of parasitological zoonotic agents, the national reference laboratory for Trichinella.

Status of the myocardium and infarct-related coronary artery in 19 necropsy patients with acute recanalization using pharmacologic (streptokinase, r-tissue plasminogen activator), mechanical (percutaneous transluminal coronary angioplasty) or combined types of reperfusion therapy.

In acute myocardial infarction, myocardial salvage is dependent on rapid restoration of blood flow. Pharmacologic (streptokinase, recombinant tissue-type plasminogen activator), mechanical (percutaneous transluminal coronary angioplasty, guide wire perforation) or combined forms of reperfusion therapy can accomplish this goal, but their effects on infarcted myocardium and vessel occlusion site have not been compared at necropsy. The heart of 19 necropsy patients who had received various forms of acute reperfusion therapy was studied: 14 had pharmacologic or combined forms of reperfusion therapy (13 streptokinase and 1 tissue-type plasminogen activator, including 4 with combined balloon angioplasty) and 5 had had purely mechanical (balloon angioplasty) reperfusion therapy. Reperfusion was initially clinically successful in all 19 patients with the average time from onset of symptoms to reperfusion being 3.7 hours. Necropsy observations separated the 19 patients into distinct subgroups based on changes in the myocardium and infarct-related coronary arteries. Of the 19 patients, 14 (74%) had hemorrhagic myocardial infarction and they all received pharmacologic or combined forms of reperfusion therapy. The remaining five patients (26%) had nonhemorrhagic (anemic) infarction and were treated with balloon angioplasty therapy alone. Increased luminal cross-sectional area was present in 8 of 9 patients with acute balloon angioplasty but severe coronary atherosclerotic plaque remained in 9 of 10 patients without acute balloon angioplasty. Severe hemorrhage surrounded angioplasty sites in all four patients who also received streptokinase or tissue-type plasminogen activator. Severe bleeding at the angioplasty site compromised the dilated coronary lumen in one patient. No patient with angioplasty alone had intraplaque bleeding. Thus, acute coronary balloon angioplasty reperfusion therapy alone appears to avoid the potentially adverse effects of myocardial and intraplaque hemorrhage while simultaneously increasing luminal cross-sectional area at the site of acute occlusion.

Squamous carcinoma of the lung in a nonirradiated, nonsmoking patient with juvenile laryngotracheal papillomatosis.

Juvenile laryngotracheal papillomatosis (JLTP)--a disease characterized by the growth of multiple, recurrent, koilocytolic papillomas of the upper respiratory tree--occurs in 1,500 to 2,000 infants and children in the United States every year. In rare instances, the papillomas, which are thought to be caused by a human papillomavirus (HPV), may extend into the distal bronchi and lungs. They are often excised surgically or by laser resection, but may regress spontaneously. Patients who receive radiation or cytotoxic drugs for this condition, or subsequently become smokers, may be predisposed to the development of bronchopulmonary carcinoma. Only six cases of bronchopulmonary carcinoma arising in persons with a history of JLTP but no history of these predisposing factors have been reported. Herein, we report the occurrence of squamous carcinoma of the left lung in a 28-year-old woman with JLTP since the age of 2 1/2 years. The carcinoma as well as randomly chosen papillomas excised over a period of years demonstrated positive nuclear staining for papillomavirus structural antigen. This is consistent with the current belief that human papillomavirus may be an important factor in the development of squamous carcinomas in various anatomic locations.

HLA-DP beta and susceptibility to multiple sclerosis: an analysis of caucasoid and Japanese patient populations.

Nonradioactive sequence-specific oligonucleotide (SSO) probes specific for the HLA-DP beta locus have been used in a simple dot-blot assay to DP beta-type samples amplified by the polymerase chain reaction (pcr) from Caucasoid (n = 24) and Japanese (n = 23) patients with multiple sclerosis (ms) as well as ethnically matched controls. In contrast to previous reports, no DP beta allele was found to be increased in either patient population. However, the results do show a dramatic difference in the allele frequencies between the two control populations, further emphasizing the need for ethnically matched controls in studies of HLA and disease.

Holoprosencephaly, ear abnormalities, congenital heart defect, and microphallus in a patient with 11q- mosaicism.

We report on a newborn male with deletion of part of 11q, the 27th reported case. Our patient had some of the clinical characteristics of the 11q- syndrome, but his male gender, liveborn status, q21 breakpoint, and mosaicism were unusual. In addition, he demonstrated holoprosencephaly, with cyclopia and arhinencephaly, manifestations previously unreported in the 11q- syndrome. We discuss the above points and review the literature on 11q-.

Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains.

The genetic background of the antimicrobial resistance of 10 selected multiresistant Salmonella serotype Typhimurium (S. Typhimurium) strains (including the emerging monophasic variant [4,5,12:i:- ]) was investigated. All strains shared class 1 integrons (with seven types of variable regions) and belonged to different lineages (L1-L6) according to their phage types, DNA polymorphisms by XbaI-pulsed-field gel electrophoresis (PFGE), integrons, and/or resistance patterns. The strains were screened for the presence and localization (chromosomal or plasmid) of 32 DNA sequences representing integron-, Tn21-like transposon-, resistance-, and virulence-plasmid genes. Strains belonging to lineage L1 (definitive phage type DT104) carried the 90-kb Salmonella virulence plasmid together with the complete or partial chromosomally located Salmonella Genomic Island 1 (SGI1). All strains belonging to the other five lineages carried their resistance determinants on various resistance plasmids. Two of these strains showed complex plasmid profiles, which included a 95 kb virulence plasmid together with two or four resistance plasmids. Two strains carried a resistance plasmid that lacked the virulence-plasmid-encoding sequences. The remaining two strains carried two different hybrid virulence-resistance plasmids. Twenty-three of the DNA sequences could be assigned to distinct XbaI genomic restriction patterns (PFGE profiles). In this way, the influence of the resistance and virulence plasmids on the PFGE profiles was determined, and several groups of resistance genes could be identified. The data obtained represent a useful epidemiological tool for tracing the emergence and distribution of multiresistant S. Typhimurium worldwide.

Detection of the induction of Salmonella enterotoxin gene expression by contact with epithelial cells with RT-PCR.

All strains of Salmonella enterica investigated were found to carry the Salmonella enterotoxin gene (stn) as determined by PCR and hybridization studies. However, when using CHO-K1 cells for testing the toxicity of the strains, not all strains showed a toxic effect (cell elongation) on the cells or did so only at a low level. The cultivation of Salmonella in contact with epithelial cells (IEC-6) led to an increase in the production of toxin. The stn gene expression was detectable with the help of the RT-PCR after 3 h of incubation. The RNA of the strains was isolated, transcribed into cDNA (with MMLV-reverse transcriptase) and amplified using PCR. The PCR products were separated electrophoretically using a polyacrylamide gel and detected by silver staining.

Phage types, antibiotic susceptibilities and plasmid profiles of Salmonella typhimurium and Salmonella enteritidis strains isolated in Istanbul, Turkey.

OBJECTIVE: To examine 13 Salmonella typhimurium and 22 S. enteritidis strains isolated from individual cases of gastroenteritis for their phage types, antibiotic susceptibilities and plasmid profiles. METHODS: The phage typing of S. typhimurium strains was done according to the method of Anderson et al, and the phage typing scheme of Ward et al was used for phage typing of S. enteritidis strains. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion method. Extended-spectrum beta-lactamase production of the strains was determined by the three-dimensional method. Plasmid profiles of the strains were examined using the method described by Kado and Liu with some modification by Graeber et al. RESULTS: Two S. typhimurium strains were DT 193 and one was DT 22, whereas 10 strains were untypable. PT 4 was the predominant phage type among S. enteritidis strains. Four S. enteritidis strains were DT 6a, three strains were PT 1 and one strain was PT 8, whereas only one strain was untypable. Eleven of 13 S. typhimurium and three of 22 S. enteritidis strains were found to be multiresistant. Ten different resistance patterns among S. typhimurium and four different resistance patterns among S. enteritidis strains were detected. Extended-spectrum beta-lactamase production was detected in 10 of 13 S. typhimurium and in three of 22 S. enteritidis strains. All S. typhimurium strains but one were found to contain at least one plasmid, with molecular masses varying between 4 and 107 MDa, and 11 different plasmid patterns were determined. Plasmid pattern analysis permitted further differentiation of the S. enteritidis strains into nine groups. A serovar-specific virulence plasmid of 36 MDa was detected in 13 of 22 S. enteritidis strains. CONCLUSIONS: The results suggest that the majority of S. typhimurium strains were closely related.

Monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methodologies.

The occurrence of antibiotic-resistant bacteria in food animals is a major public health threat. Information on the prevalence of resistance to specific drugs in both bacterial and animal species together with changes occurring over time, are necessary to understand the magnitude of the problem and to establish baselines for taking action. The aim of this paper is to define the minimum epidemiological and microbiological requirements for establishing a surveillance of antimicrobial resistance in bacteria of animal origin. Surveillance should involve different bacterial species, veterinary pathogens, zoonotic bacteria and commensal bacteria used as indicators. The collected data should be periodically updated and the reports distributed among practising veterinarians and regulatory authorities. These reports would be a useful tool for developing guidelines for the prudent use of antimicrobial agents in veterinary medicine and for action strategies.

Molecular typing methods for S. enteritidis.

The predominance of certain phage types of Salmonella enteritidis in various countries makes further epidemiological subgrouping necessary. Today this is achieved by using molecular typing methods. For various bacterial species, plasmid profiling, the pattern of outer membrane proteins and lipopolysaccharides, the fingerprinting of total genomic DNA including ribotyping, and multilocus enzyme electrophoretic typing, have proven very useful. When such methods have been applied to S. enteritidis, they revealed a homogeneous, clonal structure in contemporary PT4 isolates. Furthermore they indicate that the clone observed today emerged from a heterogeneous population before the onset of the epidemic.

Incidence of quinolone resistance in strains of Salmonella isolated from poultry, cattle and pigs in Germany between 1998 and 2001.

This paper reports the susceptibility to the quinolone nalidixic acid and the fluoroquinolone ciprofloxacin of 14,514 strains of Salmonella isolated in Germany from poultry, cattle and pigs between 1998 and 2001. Quinolone-resistant salmonellae were most frequently isolated from poultry, with a prevalence of 10.2 to 16.8 per cent. Poultry-associated serotypes, such as Salmonella Paratyphi B (d-tartrate positive), Salmonella Hadar and Salmonella Virchow, had the highest prevalence of quinolone resistance, ranging between 35 and 74 per cent. All the nalidixic acid-resistant strains also had a reduced susceptibility to ciprofloxacin, with minimum inhibitory concentrations (MICS) of 0.125 to 2 microg/ml. A comparison of the MICS for ciprofloxacin of the strains of these poultry-associated serotypes and Salmonella Enteritidis phage type 4 isolated in 1998/99 and 2000/01 indicated that there had been a shift towards higher MIC values of up to 2 microg/ml. The quinolone resistance-determining region (QRDR) of the gyrA gene and the homologue region of the parC gene of 31 selected strains were sequenced. Several different amino acid changes were observed in gyrA of the quinolone-resistant isolates at positions 83 and 87, but no substitutions were observed in parC.

Nursing staff educational preparation and patient inflicted injuries in a 160 bed psychiatric hospital.

This is a retrospective study of the incidence and severity of patient inflicted injuries upon nursing staff in a 160 bed psychiatric hospital over a period of two years. The investigation explored possible relationships between the basic educational preparation of the injured staff members, the severity of their injuries, and whether they used the behavior management techniques learned in the Mandt System classes. (The Mandt System has been used at Alaska Psychiatric Institute since 1979 to train nursing staff in how to safely deal with assaultive people.)

[Molecular biological basis for the virulence of salmonellae and the new detection methods resulting from it]. / Molekularbiologische Grundlagen der Virulenz von Salmonellen und daraus resultierende neuere Nachweisverfahren.

Molecular biological investigations on Salmonella isolates allowed the definition of several fundamental mechanisms of pathogenicity. They affect the confrontation of the pathogen with host defence mechanisms and colonization of the gut and other organs. Practical implications stem from the observation that especially the virulence plasmid carrying serotypes are able to cause systemic infections. Among them are the epidemiological relevant serotypes S. enteritidis and S. typhimurium. Our understanding of basic virulence mechanisms allows the development of new, faster and more sensitive diagnostic procedures. Among them are the polymerase chain reaction (PCR) and magnetic separation techniques using monoclonal antibodies coupled to superparamagnetic polystyrene beads. Presently both techniques are under development for commercial use for Salmonella detection as well.

Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK.

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n=356) obtained in Germany, The Netherlands and the UK (2005-2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. ß-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 ß-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.

Respiratory diseases in boid snakes are common in captivity, but little information is available on their aetiology. This study was carried out to determine the occurrence of lung associated pathogens in boid snakes with and without respiratory signs and/or pneumonia. In total, 80 boid snakes of the families Boidae (n = 30) and Pythonidae (n = 50) from 48 private and zoo collections were included in this survey. Husbandry conditions were evaluated using a detailed questionnaire. All snakes were examined clinically and grouped into snakes with or without respiratory signs. Tracheal wash samples from all snakes were examined bacteriologically as well as virologically. All snakes were euthanased, and a complete pathological examination was performed. Respiratory signs and pneumonia were detected more often in pythons than in boas. An acute catarrhal pneumonia was diagnosed more often in snakes without respiratory signs than in snakes with respiratory signs, which revealed fibrinous and fibrous pneumonia. Poor husbandry conditions are an important trigger for the development of respiratory signs and pneumonia. Different bacterial pathogens were isolated in almost all snakes with pneumonia, with Salmonella species being the most common. Ferlavirus (formerly known as ophidian paramyxovirus)-RNA was detected only in pythons. Inclusion body disease was rarely seen in pythons but often in boas. Adenovirus and Mycoplasma were other pathogens that were diagnosed in single snakes with pneumonia. In living boid snakes with respiratory signs, tracheal wash samples were found to be a useful diagnostic tool for the detection of viral and bacterial pathogens.