Quinolone resistance among Salmonella Kentucky and Typhimurium isolates in Tunisia: first report of Salmonella Typhimurium ST34 in Africa and qnrB19 in Tunisia.

AIMS: Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness. METHODS: A total of 1404 isolates of S. Kentucky (n = 1059)/S. Typhimurium (n = 345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes. RESULTS: According to our selection criteria (NAL = resistance phenotype; CIP = resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type. CONCLUSION: Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia.

Multiple cases of methicillin-resistant CC130 Staphylococcus aureus harboring mecC in milk and swab samples from a Bavarian dairy herd.

The discovery of a new mecA homolog, mecC, necessitates a modification of diagnostic procedures for the identification of methicillin-resistant Staphylococcus aureus (MRSA), as most assays used for the genotypic and phenotypic mecA detection cannot currently recognize mecC. Although the prevalence, distribution, and importance of mecC are not yet completely understood, an exchange of mecC-MRSA between humans and animals seems possible. All previously reported observations of mecC-positive strains have been sporadic. To the best of our knowledge, this is the first report about multiple cases of mecC-positive Staph. aureus in 1 dairy herd. Clonal complex 130 Staph. aureus harboring mecC were found in milk samples from 16 of 56 lactating cows kept in a herd in Bavaria, Germany. Almost all quarter milk samples positive for mecC-MRSA had the lowest possible California Mastitis Test score; composite somatic cell counts obtained from monthly milk recordings showed a mean of 51,600 cells/mL in mecC-MRSA affected cows. Additionally, mecC-positive clonal complex 130 Staph. aureus were detected in swab samples from the mammary skin and a teat lesion of 1 cow from this herd. This report suggests that mecC-carrying strains are able to spread among livestock, and that they have the ability to cause multiple cases in single herds. Therefore, future studies targeting MRSA in dairy cows need to consider mecC.

Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

Elucidation of colonization time and prevalence of thermophilic Campylobacter species during turkey rearing using multiplex polymerase chain reaction.

Two turkey flocks (male and female) and the environment of their house were investigated for the presence of thermophilic Campylobacter. Sample DNA was extracted directly from fecal material and environmental samples. Bacterial identification was done using a modified Campylobacter species specific multiplex PCR. The times needed for colonization and prevalence in male and female turkeys were determined independently. All environmental samples collected before restocking were negative in the PCR analysis, showing a good hygiene and biosecurity system. The first positive PCR results were obtained in drinking water samples at 6 d of age. Colonization occurred between the second and third week of age, starting in female birds and then followed by the males. Campylobacter jejuni was detected by multiplex PCR at first; later on, Campylobacter coli and mixtures of both were seen. After the 9 wk of age, the colonization of the flocks was completed. Great attention should be given to drinking water as a supposed source of Campylobacter contamination. Multiplex PCR proved to be a rapid, sensitive, and cheap tool for the diagnosis of Campylobacter contamination.

Bovine tuberculosis: making a case for effective surveillance.

In 2008, a cow with marked gross lesions suspicious for bovine tuberculosis (bTB) was identified by meat inspection at home slaughtering in north-western Germany. Epidemiological investigations led to the identification of another 11 affected farms with a total of 135 animals which reacted positive to the skin test. Eight affected farms had been in trade contact with the putative index farm. While the source for the initial introduction remained unknown, it was shown that all isolates tested shared the same molecular characteristics suggesting a common source of infection. The findings demonstrate that bTB can easily be transmitted via animal trade and may remain undetected for years in herds in the absence of tuberculin testing. Hence, we believe that bTB surveillance should not rely only on meat inspection, but on a combination of both meat inspection and intradermal tuberculin testing.

Mycobacterium pinnipedii: transmission from South American sea lion (Otaria byronia) to Bactrian camel (Camelus bactrianus bactrianus) and Malayan tapirs (Tapirus indicus).

Tuberculosis infections caused by Mycobacterium (M.) pinnipedii in a South American sea lion, Bactrian camel, and Malayan tapirs kept in two zoological gardens spanning a time period of 5 years are reported. The zoos were linked by the transfer of one tapir. Conventional bacteriological and molecular methods were applied to detect the pathogen. Spoligotyping and MIRU/VNTR-typing performed to assess the genetic similarity revealed identical molecular characteristics of the isolates from all animals involved. Anti-tuberculosis antibodies were detected using ELISA and a recently developed serological rapid test. The study shows that: (i) using molecular methods, the assessment of the genetic relationship of infectious agents helps to confirm the routes of infection, and that (ii) immunological tests may help to detect tuberculosis infections ante mortem more reliably and early. This would prevent the transfer of tuberculosis by asymptomatic animals.

Detection of Francisella tularensis subsp. holarctica in a European brown hare (Lepus europaeus) in Thuringia, Germany.

The isolation of Francisella tularensis subsp. holarctica biovar II (strain 06T0001) from a European brown hare (Lepus europaeus) from Thuringia, Germany, is described for the first time. Identification of the microorganism was carried out by phenotypic characterisation, partial sequencing of the 16S rRNA gene and specific PCR using the primers TUL4-435/TUL4-863 and FtC1/FtC4. The epidemiology of tularemia in Germany is discussed and a risk assessment for humans is made.

Microarray-based characterisation of a Panton-Valentine leukocidin-positive community-acquired strain of methicillin-resistant Staphylococcus aureus.

Recent years have witnessed the emergence of novel methicillin-resistant Staphylococcus aureus (MRSA) strains that produce the potent toxin Panton-Valentine leukocidin (PVL). PVL-positive strains can cause complicated skin infections or necrotising pneumonia with high mortality, and these strains have the potential for epidemic spread in the community. In 2004-2005, two case clusters and two isolated cases were observed in eastern Saxony and southern Brandenburg. These were the first known infections with PVL-positive community-acquired MRSA (caMRSA) in this part of Germany. The isolates belonged to agr type III, spa type 44 or spa type 131, and showed a SmaI macrorestriction pattern that corresponded to caMRSA of clonal group ST80. The isolates were susceptible to levofloxacin, macrolides, clindamycin, gentamicin and vancomycin. Most isolates showed resistance to tetracycline and fusidic acid because of the presence of the tetK and far1 genes. A novel plasmid (designated pUB102) harbouring far1, tetK and blaZ was characterised and partially sequenced. Microarray analysis revealed that the caMRSA isolates harboured genes encoding several bi-component toxins (lukF/S-PVL, lukD/E, lukS/F plus hlgA, and another putative leukocidin homologue). Neither tst1 nor genes for enterotoxins A-Y were detected, but the isolates harboured several staphylococcal enterotoxin-like toxin genes (set genes), as well as genes encoding an epidermal cell differentiation inhibitor (edinB) and exfoliative toxin D (etD). Comparative analysis of other isolates from Australia, Germany, Switzerland and the UK showed that these isolates were representative of a widespread clone of caMRSA.

The use of DNA microarray technology for detection and genetic characterisation of chlamydiae.

Due to its highly parallel approach, DNA microarray technology opens up new possibilities that may be particularly beneficial for laboratory diagnosis of infectious diseases. We developed a microarray assay for detection and differentiation of all currently defined chlamydial species belonging to the genera Chlamydia and Chlamydophila using the ArrayTube system, which we found to be particularly user-friendly and economical. The test includes PCR amplification of a 23S rDNA target region with concurrent biotinylation and subsequent hybridisation in the ArrayTube, a micro-reaction tube carrying the microarray chip on the bottom. In addition to high specificity, the assay was shown to allow detection and genetic characterisation of single PCR-amplifiable target DNA copies.

[Comparative studies on detection of Chlamydophila psittaci and Chlamydophila abortus in meat turkey flocks using cell culture, ELISA, and PCR]. / Vergleichende Untersuchungen zum Nachweis von Chlamydophila psittaci und Chlamydophila abortus in Putenmastbetrieben mittels Zelikultur, ELISA und PCR.

The prevalence of chlamydia in 10 meat turkey flocks was investigated. As samples served of each moment of collection and sex of the animals 10 cloacal swabs which were taken at the age of 1, 4, 8 and 12 (females) or 16 weeks (males) and at the time of slaughter at the age of 16 or 20 weeks. Spleen samples were taken at the time of slaughter, additionally. These were pooled making 1 pool out of 5 individual samples. The cloacal and spleen pools were examined by nested PCR (nPCR), Capture-ELISA and Capture Blocking-ELISA directly as well as after isolation attempts in cell cultures. The most sensitive method to detect chlamydia, with 6 isolates proved to be the isolation by cell culture followed by detection using nPCR. Not corresponding to the results of the nPCR were 4 positive reactions found by the Capture-ELISA which could in no case be affirmed by Capture-Blocking-ELISA. The direct examination of cloacal swab pools by nPCR proved positive in only 2 cases. In contrast to this the examination of these samples by Capture-ELISA showed a high percentage of 71.9% positive results, of which only 2 cases were confirmed by nPCR and none by Capture-Blocking-ELISA. Of the 8 Chlamydia positive results in the nPCR 7 could be classified by DNA sequencing to Cp. abortus and only one to Cp. psittaci.

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses.

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.

Evidence of infection in tortoises by Chlamydia-like organisms that are genetically distinct from known Chlamydiaceae species.

Nasal lavage fluid was collected from 155 tortoises, mostly Testudo spp., that were kept as companion animals and suffered from nasal discharge. Examination for chlamydial DNA by PCR assays targeting the ompA, ompB, and groESL genes, as well as the 16S rRNA signature region and the 16S-23S intergenic spacer, respectively, revealed 16 (10.3%) positive animals. Sequence analysis of PCR products indicated high homology to the family Chlamydiaceae. Phylogenetic trees constructed from partial sequences of the ompA and 16S rRNA genes showed that the present samples clustered outside the nine species of Chlamydia and Chlamydophila. Sequences of the nearest relative, Chlamydophila pecorum, were still clearly distinct from those of the positive tortoise samples. This suggests that the tortoises had been infected by Chlamydia-like agents, the taxonomic identity and pathogenic importance of which has yet to be established.

Genetic characterization of a Chlamydophila pneumoniae isolate from an African frog and comparison to currently accepted biovars.

The amphibian isolate DE177 identified as Chlamydophila (C.) pneumoniae was sequenced in five genomic regions: 16S ribosomal RNA gene, 16-23S intergenic spacer, ompA, ompB, and groESL genes. Comparison with corresponding sequences of the currently accepted equine, human and koala biovars of C. pneumoniae revealed that koala strains represented the most closely related taxon, although sequence dissimilarities in the ompA (VD4) and ompB gene regions were noted. In this respect, the present isolate is distinct from a previously described frog isolate (Berger et al., 1999) whose sequence analysis yielded identity to the koala biovar. As three of the nucleotide substitutions in ompA (VD4) of DE177 will be translated into two altered amino acids the possible existence of another biovar is discussed.

A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster.

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1 /P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis.

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.

Neospora caninum and Waddlia chondrophila strain 2032/99 in a septic stillborn calf.

Characteristics of an intracellularly growing micro-organism isolated from an aborted bovine foetus are described. The organism replicated within cytoplasmic vacuoles, was resistant to penicillin and exhibited structural characteristics compatible with Waddlia chondrophila. An ELISA specific for Chlamydia spp., immunofluorescence tests using antibodies directed against Chlamydia spp. or Simkania negevensis, and PCR using Chlamydia-specific primers showed that the agent was distinct from Chlamydiae or S. negevensis. Determination of 16S and partial 23S ribosomal RNA gene sequences in combination with the PCR results and the morphological, antigenic and developmental characteristics provided evidence that the isolate 2032/99 can be classified as W. chondrophila or a closely related organism.

Detection of Mycoplasma bovis using in vitro deoxyribonucleic acid amplification.

Detection of Mycoplasma bovis by traditional culture methods is rather time-consuming and is often hampered by bacterial contamination. The development of a rapid and specific alternative is, therefore, an important prerequisite in improving the diagnosis of bovine diseases caused by this agent. The authors have successfully used nucleic acid probes containing genomic restriction fragments of M. bovis cloned into the plasmid vector pUC19 for species-specific detection by dot blot hybridisation of small quantities of M. bovis deoxyribonucleic acid (DNA). The problem of direct M. bovis detection from contaminated milk could not be solved using this procedure. Therefore, further research was conducted using in vitro DNA amplification by polymerase chain reaction (PCR). Species-specific nucleic acid probes were sequenced and suitable PCR primers selected. Using the PCR procedure, ten colony-forming units (CFU) were detected from broth cultures and, after DNA isolation, the equivalent of 1 CFU was detected. Direct detection of M. bovis from biological samples proved extremely difficult due to protein interference. It was shown, however, that direct PCR detection from milk is possible after effective protein removal by combined extraction and protease digestion.

Comparison of various diagnostic methods for the detection of Mycoplasma bovis.

Mycoplasma bovis, the main causative agent of mycoplasmal mastitis, arthritis and pneumonia in cattle, causes considerable economic losses. Veterinary hygiene measures would be most effective if introduced at an early stage, especially the culling of cows shedding the pathogen for the control of mastitis. It is therefore crucial to ensure that diagnostic methods are available which can perform rapid and specific detection of the agent at acceptable costs. Six different detection methods have been compared and evaluated in terms of performance parameters and suitability for routine diagnosis. Conventional M. bovis isolation and identification from culture is the only technique used for routine diagnosis at present. However, this process is rather laborious and time-consuming, and final results are available only after several days. Enzyme-linked immunosorbent assay (ELISA) techniques can be used to screen for M. bovis antibodies or antigens in clinically-diseased animals. Detection of the agent in subclinical cases was accomplished in pre-incubated samples by an antigen capture ELISA involving a monoclonal antibody. Whole-cell protein patterns generated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to identify and classify field isolates. Nucleic acid hybridizations using probes of defined specificity were conducted both as filter dot blot assay and in solution with ribosomal ribonucleic acid as the target. The latter was found to be potentially suitable for the screening of biological samples, although problems due to high background and reduced specificity remained. Finally, the presence of M. bovis cells in culture supernatant and in milk samples was demonstrated using the polymerase chain reaction. This procedure is potentially superior to all others currently available, due to its high sensitivity, specificity and speed. However, a number of practical problems must be solved prior to full-scale introduction of this technique for routine diagnosis.

[Distribution, serovar affiliation and epidemiologic behavior of Cryptococcus neoformans isolates from ornamental bird breeds]. / Verbreitung, Serovarzugehörigkeit und epidemiologisches Verhalten von Cryptococcus-neoformans-Isolaten aus Ziervogelzuchten.

Investigations of faeces samples from breeding stocks of companion birds in the federal state of Thuringia revealed a high contamination rate of companion birds with Cryptococcus (Cr.) neoformans var. neoformans. The prevalence of Cr. neoformans var. neoformans correlated with the spectrum of bird species present in the respective breeding units. The causes for that are not clear at the moment. Sensitivity of Cr. neoformans var. neoformans towards alkaline agents was not confirmed and was ruled out as a reason for different tenacity of the yeast in various bird breedings. Differentiation of varieties within Cr. neoformans was possible on the basis of proline assimilation, determination of canavanine resistance, EDTA urease test, as well as Cr. neoformans var. neoformans factor sera and PCR fingerprinting. Serological differentiation of serovars and PCR fingerprinting resulted in subdivision of Cr. neoformans var. neoformans isolates into two groups, which corresponded to serovars A and D. A prevalence of serovar A isolates was found in investigated bird breeding stocks. This also corresponded to the distribution of Cr. neoformans var. neoformans described in literature in humans with cryptococcosis in Germany. Consequently, serovar A or D infections of patients may be connected with their contacts to Cr. neoformans-excreting companion birds.

[Identification and differentiation of Campylobacter fetus subspecies by PCR]. / Identifizierung und Differenzierung der Campylobacter-fetus-Subspezies mittels PCR.

The species Campylobacter (C.) fetus is divided into the subspecies venerealis and fetus, which differ in epidemiology and clinical importance. The differences between these subspecies make an accurate distinction essential. Differentiation of C. fetus by traditional microbiological methods is only based on two reactions (tolerance to glycin, Na selenite reduction), in which C. fetus ssp. venerealis reacts negatively. However, the value of both reactions is limited. We used a specific PCR-based assay for identifying and differentiating the two C. fetus subspecies, which was recently developed by HUM et al. (1997). In this assay, a 764 bp amplicon is produced using primers MG3F and MG4R for both subspecies of C. fetus. In contrast to HUM et al. (1997), this amplicon was approximately 200 bp smaller. This discrepancy can't be explained. Afterwards, the primers VenSF and VenSR are used for differentiation. The identification of the sub-species venerealis is based on the presence of a 142 bp amplicon, which is not formed with subspecies fetus. The type strains of both C. fetus subspecies were used as positive controls. Non-specific reactions were not observed. In this PCR assay, 73 field strains were investigated (among them 24 C. fetus ssp. veneralis, 26 C. fetus ssp. fetus). In these investigations, the method has proved its diagnostic suitability. The results of the traditional microbiological differentiation of the C. fetus field strains could be confirmed by the PCR assay. In future, the traditional phenotypic characterization of C. fetus subspecies remains indispensable, but this PCR assay constitutes a valuable method for the confirmation of these results.