Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5′ Taq nuclease assay: a new tool for diagnosing infectious coryza

A 5′ Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5′ Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5′ Taq nuclease assay was demonstrated.     

Quinolone resistance among Salmonella enterica and Escherichia coli of animal origin

Escherichia coli and Salmonella enterica are major pathogens of worldwide importance in the animal industry. Antimicrobial therapy is an important tool in reducing the enormous losses caused by these infectious agents, however, resistance to existing antimicrobials especially quinolones and fluoroquinolones is widespread and of concern to veterinarians. Iranian isolates of E. coli and S. enterica from different species of animals were examined for resistance to quinolones and fluoroquinolones. Thirty-five S. enterica isolates were recovered from different animal origins. Twenty-five E. coli strains were also isolated from poultry with colibacillosis. Eleven strains of E. coli were isolated from cloacal swabs from healthy chicken. All of the E. coli strains were identified by biochemical tests. Gene invA was detected in all of the Salmonella isolates. Serogroups of Salmonella were determined by colored rapid latex test. There was no relationship among serogroups of Salmonella and resistance to quinolones. In vitro antibiotic activities of four antibiotic substances against the isolates were determined by disk diffusion test. Forty percent of S. enterica and 96% of E. coli were found to be resistant to nalidixic acid. Fifty-six percent, 72%, and 36% of E. coli strains were resistant to ciprofloxacin, enrofloxacin, and norfloxacin, respectively. However, 11.42%, 22.85%, and 5.71% of Salmonella strains were resistant to ciprofloxacin, enrofloxacin, and norfloxacin, respectively.     

Resistance Patterns of Streptococcus pneumoniae from Children in Central Italy

 Nasopharyngeal swabs were collected from children aged 3–5 years in central Italy who were attending day-care centres or hospital outpatient clinics. One hundred and twenty-one strains of Streptococcus pneumoniae isolated were tested for susceptibility to penicillin, cefotaxime, erythromycin, clindamycin, tetracycline, chloramphenicol and cotrimoxazole. A high prevalence of penicillin-resistant (14%), erythromycin-resistant (60%) and multiply resistant strains (53%) were found. An unusual finding was that 49 of the 64 (76.6%) multiply resistant strains were penicillin-susceptible, 28 serogroup 6 strains also being resistant to the other antibiotics tested. Such strains have not previously been reported from Italy but have the same features as strains recently found in child carriers in the eastern Mediterranean area.     

Pseudo-Outbreak of Cupriavidus pauculus Infection at an Outpatient Clinic Related to Rinsing Culturette Swabs in Tap Water

Cupriavidus pauculus is a water microorganism rarely isolated from clinical specimens. We describe a pseudo-outbreak in which multiple strains that were associated with moistening of culturette swabs with tap water were isolated from a single clinic before collecting the patient specimen.     

Infant colonization by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>: role of maternal carriage

Infant colonization by Staphylococcus aureus has not been adequately investigated. In this study, we aimed to define determinants associated with the carriage of S. aureus in early infancy. Serial nasal swabs were collected from 128 infants and their mothers at months 0, 6, and 12 postpartum. S. aureus isolates were characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, and the presence of chromosomal mecA and of Panton–Valentine leukocidin (PVL) genes. S. aureus was isolated in 17.7% and 15.7% of swabs from infants and mothers, respectively. Carriage rates were higher in infants with carrier mothers, non-smoking mothers, and many siblings. Persistent carriage rates were higher in infants with carrier or non-smoking mothers. S. aureus typing revealed identical strains in 10/15 investigated infant–mother pairs. Among 19 investigated S. aureus isolates from infants, ten harbored mecA and two harbored PVL genes, and these determinants were concomitantly present in isolates from mothers. Resistance to methicillin was 43.6% among all isolates from infants. In conclusion, isolates from infants were commonly identical to isolates from their mothers, pointing to a principal role of maternal carriage in S. aureus colonization in infants.     

Characterization of Staphylococcus aureus strains isolated from humans in Argentina

Humans are a natural reservoir of Staphylococcus aureus and asymptomatic colonization is far more common than infection. The aim of this work was to characterize genotypically 68 S. aureus strains isolated from nasal swabs of healthy people and from human clinical infections. A total of fourteen (20%) strains were susceptible to all the antimicrobials tested. The strains isolated from nasal swabs showed the lowest percentages of resistance. Resistance to one or more than one antibiotics tested was detected in 83% and 70% of the S. aureus strains isolated from clinical infections and nasal swabs, respectively. All of the 68 S. aureus strains were subject to RAPD-PCR analysis. Cluster A-I grouped 42 (87%) clinical infection strains and cluster A-II grouped 13 (65%) strains isolated from nasal swabs suggesting a genetic relationship among S. aureus strains. Cluster A-II grouped 65% of the S. aureus strains associated with the anterior nares, suggesting that these strains may be adapted to this site. Furthermore, five RAPD profiles isolated from nasal swabs, belonged to clusters B to F, were similar to strains isolated from clinical infection, suggesting that they might have a high propensity to cause disease. The results of the present study allow a characterization of S. aureus strains isolated from humans and shows that some S. aureus genotypes from nasal swabs are similar to the genotypes obtained from clinical infections, suggesting that clinical isolates may be originated from human normal flora.     

Thymidine kinase-negative bovine herpesvirus type 1 mutant is stable and highly attenuated in calves

Summary Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk^–) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk⁺ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk^– BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk^– BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk^– BHV-1 vaccination reduced the multiplication of tk⁺ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk^– BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without revertingin vivo to tk⁺. The results demonstrate that the tk^– BHV mutant is attenuated and efficacious as a vaccine.With 1 Figure     

Vannella sp. harboring Microsporidia-like organisms isolated from the contact lens and inflamed eye of a female keratitis patient

Viable Hartmannella sp. and two strains of Vannella sp. – but no Acanthamoebae– multiplied on NN-agar inoculated with pieces of the contact lens from a female keratitis patient. Within the cytoplasm of one Vannella isolate, intracellular parasites could be observed whose earliest stages were developing within the nucleus, resembling those Microsporidia-like parasites seen within Vannella isolated recently from a warm tapwater system. This assumption was also confirmed by electron microscopy. In swabs taken directly from the cornea, Pseudomonas aeruginosa were identified, but they did not yield any growth of amebas in culture. However, cocultivation of parasite-free Vannella strains with the above-mentioned swab matter resulted in infected amebas harboring the same intracellular parasites seen before. This infection could be established only if the corresponding spores were present as infective agents in the swab matter. The successful treatment of the patient with antibiotics supports the assumption that P. aeruginosa was the main cause of the corneal ulceration. The extent to which the Microsporidia-like organisms may have been involved in the development of keratitis remains a matter of discussion. Received: 17 October 1999 / Accepted: 10 November 1999     

Methicillin‐resistant and vancomycin‐intermediate Staphylococcus aureus colonizing patients and intensive care unit environment: virulence profile and genetic variability

This study aims to determine the prevalence of Staphylococcus aureus colonizing patients and ICU environment of a teaching hospital, the virulence and antimicrobial susceptibility profile of the isolates, and to evaluate the genetic relationship among them. A total of 536 swabs (134 of patients and 402 of ICU environment) were collected and analyzed to detect S. aureus. The antimicrobial susceptibility of the isolates was determined by disk diffusion test, and the detection of the mecA and virulence factors genes was performed by PCR, in addition to SCCmec typing. The genetic similarity of the isolates was determined by PFGE. Staphylococcus aureus was isolated in 12.7% of the swabs. The prevalence of colonization was 13.4% in patients and 12.4% in the environmental samples. The multidrug resistance was determined in 82.4% of the isolates. The prevalence of methicillin‐resistant S. aureus was 20.6%, with 50.0% classified as SCCmec IV. The intermediate resistance to vancomycin was detected in 5.9% and 4.4% of the isolates obtained from patients and environment, respectively. Identical isolates obtained from different patients and sources were grouped into several clusters. The results showed dissemination of multidrug‐resistant strains between patients and fomites and the persistence of MRSA and VISA isolates in the ICU environment.     

Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru

Pathogenic strains of Acanthamoeba genus are the causative agents of fatal granulomatous amoebic encephalitis and a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. In a previous study, Acanthamoeba strains were isolated from nasal swabs collected from healthy individuals in Peru. In the present study, the pathogenic potential of the isolated strains was established based on temperature and osmotolerance assays as well as the secretion rate of extracellular proteases. Based on these experiments, four strains that showed the highest pathogenic potential were selected for sensitivity assays against two molecules (voriconazole and chlorhexidine) which are currently used for the treatment of Acanthamoeba infections. After performing sensitivity and activity assays, it was found that both drugs were active against the tested strains. However, voriconazole showed higher activity against the studied strains compared to chlorhexidine. Therefore, voriconazole should be established as a first-line treatment against Acanthamoeba infections at least in the studied region of Peru.     

Occurrence of Hsw1N1 subtype influenza A viruses in wild ducks in Europe

Summary Two identical strains of influenza A viruses antigenically related to swine influenza (Hsw1N1) have been isolated from adult mallard ducks(Anas platyrhynchos) in Southern Germany. They were designated A/Duck/Bavaria/1/77 and A/Duck/Bavaria/2/77. Serologic tests revealed a close antigenic relationship to the strain A/Duck/Alberta/35/76.Experimental infections of piglets with strain A/Duck/Bavaria/1/77 demonstrated the susceptibility of swine to this virus strain. The virus was isolated from nasal swabs of infected piglets up to 8 days p. inf. and from contact animals up to 9 days. No seroconversion was detected during an observation period of 30 days.     

Further evidence of the circulation of PMV-4 and influenza viruses with N2-1957 enzyme in the migratory waterfowls

In the years 1980-1984, one paramyxovirus type 4 and 11 influenza viruses were isolated from cloacal swabs collected from migratory waterfowls in Fed. Rep. Germany. One influenza virus of H4N8 subtype was isolated from swabs of commercial ducks collected at an abbatoir. Seven of 10 influenza strains, isolated from mallard ducks and coot were identified as a mixture of 2-3 strains of H1, H4, and H5 subtype; 3 virus strains from the same locality relate antigenically to subtype H4 with enzyme serologically identical with N2--Singapore/57 as demonstrated by means of polyclonal and monoclonal antibody.     

Corynebacterium macginleyi Has to Date Been Isolated Exclusively from Conjunctival Swabs

Fifteen strains of Corynebacterium macginleyi were exclusively isolated from conjunctival swabs of patients with either conjunctivitis or corneal ulcers. Up to now, only three C. macginleyi strains had been described in the literature. The characteristics of the 15 patients from whom C. macginleyi was isolated are outlined, characteristics useful for the identification of C. macginleyi are described, and the antimicrobial susceptibility pattern of the species is provided. C. macginleyi is uniformly susceptible to penicillins, quinolones, and aminoglycosides. Although considered to be of rather low pathogenicity C. macginleyi seems to have the potential to cause superinfections in selected patients with ocular surface problems.     

Phenotypical and Genotypical Characterization of Neisseria meningitidis Carrier Strains Isolated from Polish Recruits in 1998

 The aim of this study was to investigate the Neisseria meningitidis carriage rate among two cohorts of Polish recruits upon entry to the military and during the first 2 months of their service, i.e. in the spring and autumn of 1998, and to characterize the meningococcal strains isolated. Pharyngeal swabs were taken four and five times from 151 and 168 men, respectively. Altogether, 81 and 180 meningococcal isolates representing 54 and 102 different strains were recovered. The overall rates of carriage in the spring and in the autumn were 36% and 61%, and, among recruits who submitted to sampling on at least three occasions, 39% and 55%. Eighty-three of 156 (53%) meningococcal carrier strains were nongroupable; among the remaining strains, serogroup B was predominant (32% of all carrier strains). In both surveys the predominant phenotype was Neisseria meningitidis NG : 21 : P1.7.     

Detection ofBordetella pertussis by determination of adenylate cyclase activity

The adenylate cyclase activity ofBordetella pertussis in clinical isolates was measured in calmodulin-supplemented Stainer-Scholte broth by the rate of conversion of ATP to cyclic AMP. Analysis of 250 stock strains ofBordetella pertussis showed that measurable adenylate cyclase activity was produced by all strains. In clinical testsBordetella pertussis was isolated from 135 (22 %) of 605 swab samples. Increased adenylate cyclase activity was detected in 124 (92 %) Stainer-Scholte broth cultures of these samples. A total of 475 swabs contained other bacteria or had no growth; only one of the Stainer-Scholte broth cultures of these swab samples contained measurable adenylate cyclase activity. The results indicate that testing for adenylate cyclase activity provides a specific and sensitive means for detectingBordetella pertussis in clinical specimens.     

Epidemiology of bacteria and viruses in the respiratory tract of humans and domestic pigs

Bacteria and viruses were analysed in the upper respiratory tract of symptomatic pig farmers and their domestic pigs. Eighty six human nasal and 495 (50 pools) porcine snout swabs were collected in Schleswig-Holstein, Germany. Staphylococcus (S.) aureus (62.8%, 54/86), human rhino- and coronaviruses (HRV, 29.1%, 25/86; HCoV, 16.3%, 14/86) were frequently detected in humans, while Haemophilus parasuis (90.0%, 45/50), Mycoplasma hyorhinis (78.6%, 11/14), Enterovirus G (EV-G, 56.0%, 28/50) and S. aureus (36.0%, 18/50), respectively, were highly prevalent in pigs. The detection of S. aureus in human follow-up samples indicates a carrier status. The methicillin-resistant phenotype (MRSA) was identified in 33.3% (18/54) of nasal swabs and in one of 18 (5.6%) pooled snout swabs that were tested positive for S. aureus. Strains were indicative of the livestock-associated clonal complex CC398, with t011 being the most common staphylococcal protein A type. Enterobacterales and non-fermenters were frequently isolated from swabs. Their detection in follow-up samples suggests a carrier status. All were classified as being non-multiresistant. There was no example for cross-species transmission of viruses. In contrast, transmission of S. aureus through occupational contact to pigs seems possible. The study contributes to the ‘One Health’ approach.     

Pathogenicity and kinetics of virus propagation in swine infected with the cytopathogenic classical swine fever virus containing defective interfering particles

Summary.  To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E⁺ strain), WB82 and WB82/E⁺ strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E⁺ strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E⁺ strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV. Received January 14, 2002; accepted August 19, 2002     

Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for control of MRSA nosocomial transmission. We aimed to evaluate the performance of the GeneXpert real-time PCR system using the Xpert MRSA assay on a collection of 40 representative Belgian MRSA strains and for MRSA screening of geriatric inpatients. Double nasal swabs were used: the first swab for the Xpert MRSA assay and the second for culture onto chromogenic selective medium and enrichment broth. All but 1 of the 40 collection strains were recognized as MRSA by the Xpert MRSA assay. Nares swabs were prospectively collected from 246 inpatients including 25 nasal MRSA carriers. Compared with enriched cultures, the sensitivity, the specificity, and the positive and negative predictive values of the Xpert MRSA assay were 69.2%, 97.7%, 78.3%, and 96.3% respectively. The 7 evaluable false-negative results according to the assay were due to its possible lack of sensitivity (n = 3) and to the occurrence of a Belgian MRSA clone carrying a particular staphylococcal chromosomal cassette mec (SCCmec) type IV variant (n = 4) not targeted by the current Xpert MRSA assay. Because of the evolution of SCCmec in MRSA, new primers should be designed and further studies are warranted to ensure continuous monitoring of this assay.     

Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts

In this paper, the infectivity for mice of Trypanosoma cruzi I and II strains isolated from sylvatic animals, triatomines, and humans is determined using fresh blood examination, hemoculture, culture of macerated organs, and polymerase chain reaction (PCR). Six strains were considered to have low infectivity (9.1–18.2%), five medium (27.3–45.4%), and one high (100.0%). Infectivity of T. cruzi strains isolated from sylvatic animals was significantly higher than that of strains isolated from humans and triatomines (p=0.0141). No significant difference was observed between the infectivity of T. cruzi I and II strains. The parasite was detected by fresh blood examination in one strain, by hemoculture and culture of macerated organs in four strains, and by PCR in all strains. We conclude that the infectivity is related to the host from which the strains were isolated, but the infectivity is not related to the genetic group of the parasite. We also conclude that the majority of the strains studied have low and medium infectivity for mice, and that PCR is an important tool to detect T. cruzi in strains with this biological characteristic.     

Positive predictive value of the Xpert MRSA assay diagnostic for universal patient screening at hospital admission: influence of the local ecology

The purpose of this study was to assess the accuracy of the Xpert MRSA assay (XP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission. Nasal swabs were prospectively collected for MRSA screening from 1,891 patients admitted to a teaching hospital. XP results were compared to chromogenic agar culture results. MRSA was cultured in 61 specimens (3%). Compared with culture, XP had a sensitivity, specificity, positive, and negative predictive value of 60.7, 97.3, 37.8, and 98.9%, respectively. The median turnaround time (TAT) for the results was 3 h. Of 24 MRSA isolated from XP-negative samples, three harbored composite SCCmec. Among 61 samples with culture-negative but XP-positive results, 15 methicillin-susceptible S. aureus (MSSA) isolates tested positive by XP on pure colony lysates. These MSSA included: (i) strains with SCCmec deletion encompassing mecA and (ii) multilocus sequence typing (MLST) clonal complex (CC) 1 strains harboring a chromosomal sequence homologous to one of the orfX–SCCmec junction sequences targeted by XP. On account of the low sensitivity and positive predictive value in a hospital patient population with moderate prevalence of MRSA, culture still appears to be necessary in order to confirm polymerase chain reaction (PCR) results. The emergence of new SCCmec variants and the presence of MSSA harboring cross-reactive SCCmec-like elements may challenge the successful implementation of such detection systems.