Pathologic findings and causes of death in bottlenose dolphins Tursiops truncatus stranded along the Georgia coast, USA (2007-2013).

Between 2007 and 2013, before the 2013 cetacean morbillivirus outbreak, 26 fresh bottlenose dolphin carcasses were necropsied on the coast of Georgia, USA. Here, we present the pathological and microbiological findings associated with their most likely causes of death. The primary cause of death was determined in 25 individuals and included systemic bacterial infection (n = 7), verminous and bacterial bronchopneumonia (n = 5), drowning/entanglement (n = 5), disseminated histoplasmosis (n = 1), intestinal intussusception (n = 1), vegetative endocarditis (n = 1), meningitis (n = 1), necrotizing dermatitis (n = 1), disseminated angiomatosis (n = 1), emaciation (n = 1) and stingray spine trauma (n = 1). Histiocytic and eosinophilic bronchopneumonia associated with Halocerchus sp. infection was observed in 69% of the animals (18/26) and eosinophilic gastritis due to Anisakidae nematodes was found in 36% of the examined stomachs (8/22). Moderate to severe eosinophilic pancreatitis with fibrosis was observed in 4 animals infected with Brachycladiidae trematodes. Proliferative and ulcerative lymphoplasmacytic dermatitis was found in 5 animals and was considered to contribute to deteriorated health status in 2 calves. Pulmonary and lymph node angiomatosis were observed in 15 and 10 animals, respectively. In at least 2 animals, the concentration of polychlorinated biphenyls (PCBs) in the blubber exceeded 1500 µg g-1 of lipid. Bottlenose dolphins stranded on the Georgia coast have a wide range of inflammatory lesions associated with a variety of helminth, bacterial, and fungal pathogens. Some resident animals have also been exposed to high levels of PCB contamination, which could reduce host immunocompetence. Higher exposure to these or other pathogens could result in further decline in the health of resident and migrant dolphin populations in this region.

Pneumococcal serotypes causing pediatric meningitis in Turkey: application of a new technology in the investigation of cases negative by conventional culture.

The surveillance of serotypes causing invasive pneumococcal disease (IPD) provides further insight into the pathogenesis of pneumococcal disease and is important in order to track vaccine impact. Although the Quellung reaction has been accepted as the standard method for serotyping, prior antibiotic use causes a gap in studies based on bacterial culture. A total of 31 cerebrospinal fluid (CSF) samples found to be positive for Streptococcus pneumoniae by polymerase chain reaction (PCR) targeting the ply gene during an active surveillance were tested in a Bio-Plex multiplex antigen detection assay capable of detecting 14 serotypes/groups (1, 3, 4, 5, 6A, 6B, 7F/A, 8, 9V, 14, 18, 19A, 19F, and 23F). Twenty-seven CSF samples could be serotyped. The most common serotypes were serotypes 5 (n = 7), 19F (n = 5), 1 (n = 3), and 23F (n = 3). Theoretical coverage rates by the heptavalent (PCV7), 10-valent (PCV10), and 13-valent (PCV13) pneumococcal conjugate vaccines for bacterial meningitis were 48.1, 85.2, and 92.3%, respectively, for all age groups and 71.4, 85.7, and 100.0%, respectively, for those under 2 years of age. We propose that antigen detection assay used in conjunction with a PCR assay can be effectively applied in CSF samples to detect the pneumococcal serotypes, especially when the patient may have already been treated and, therefore, the cultures would be negative.

Antimicrobial susceptibility testing of historical and recent clinical isolates of Bordetella pertussis in the United Kingdom using the Etest method.

Reports of the development of antimicrobial resistance by Bordetella pertussis to macrolides in the United States and Taiwan, together with a recent increase in pertussis notifications and laboratory-confirmed cases in England and Wales in 2008, prompted the examination of historical and recent clinical isolates from patients for evidence of such resistance in our collection. Isolates submitted to our laboratory as part of the enhanced surveillance scheme for pertussis, from 2001 to 2009, were tested against three agents, erythromycin, clarithromycin and azithromycin, by the Etest (bioMérieux) method. All isolates (n = 583) were fully susceptible to all three agents tested (minimum inhibitory concentrations [MICs] 256 microg/ml. Although no evidence of resistance was found in the strains tested from the United Kingdom, screening for antimicrobial resistance of B. pertussis may be warranted in cases that are unresponsive to macrolide treatment and to provide early warning of such emergence in the future.

On the mechanism for the red-cell accumulation of mefloquine, an antimalarial drug.

The accumulation of the antimalarial drug mefloquine by human red blood cells has been studied by 19F-NMR spectroscopy. The uptake process was nonlinearly dependent on the external drug concentration. Concentrations inside cells as high as 60-times greater than those in the extracellular phosphate-buffered-saline were observed. Red-cell ghosts were also found to accumulate mefloquine with high-affinity binding sites for the drug. Hemoglobin was found to bind mefloquine with low affinity, but due to the high concentration of this protein it is a significant drug compartment in the red cell. Analysis of the 19F-NMR chemical shifts and linewidths of mefloquine in the presence of red cells, red-cells ghosts and hemoglobin indicates restricted mobility of the drug in the membrane-bound state and slow exchange with the extracellular medium. This is a significant characteristic of the reaction in connection with the prophylactic activity of the drug. Exchange of the drug between hemoglobin and the red-cell membrane, however, is fast and may play an important role in the bioavailability of the drug to the parasite.

Subunit structure and dissociation of Callinectes sapidus hemocyanin.

The hemocyanin of the blue crab, Callinectes sapidus has two major components sedimenting with approximate sedimentation coefficients of 17 S and 25 S. Molecular weight data based on light scattering and sedimentation equilibrium measurements at pH 7.8 suggest that the two components have molecular weights of approximately 450 000 and 900 000 in the presence of stabilizing Ca2+. In the absence of Ca2+, the molecular weights are found to be about 5% lower, suggesting some dissociation of the hemocyanin components. Circular dichroism and optical rotatory dispersion measurements in the far-ultraviolet region gave nearly identical spectra for the two components. Based on the reference parameters of Chen et al. (Chen, Y.H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131 and Chen, Y.H., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3340--3359), estimates of 16--20% alpha-helix, 40--60% beta-structure, and 30--40% random organization were obtained for the two hemocyanin components. Exposure to 6 M Gdn HCl gave light scattering molecular weights of approx. 68 000 and 77 000, which is close to one-sixth of the molecular weight of the 17 S component. These results support the view that the two components of C. sapidus hemocyanin share the hexameric and dodecameric organization common to arthropod hemocyanins. The salts of the Hofmeister series and the ureas are found to dissociate the dodecameric component with the former exhibiting the usual order of effectiveness of NaCl, NaBr, NaI, and NaClO4 dissociation, while the ureas show an inverse order of decreasing effectiveness in going from urea to methyl-, ethyl- and propylurea. This suggests that polar and ionic interactions are relatively more important than hydrophobic interactions for the stabilization of the dodecameric form of C. sapidus hemocyanin. The dissociation behavior of the 17 S hexameric species by GdnHCl in the 0--1.5 M concentration region (where essentially no denaturation occurs), based on light scattering molecular-weight measurements, is satisfactorily accounted for by equations describing the dissociation of hexamers to monomers.

Invasive group A streptococcal disease: should close contacts routinely receive antibiotic prophylaxis?

Group A streptococci (Streptococcus pyogenes) causes a wide range of illnesses from non-invasive disease--eg, pharyngitis--to more severe invasive infections--eg, necrotising fasciitis and toxic shock-like syndrome. There remains uncertainty about the risk of secondary cases of invasive disease occurring among close contacts of an index case and how best to manage that risk. We do not consider that currently available evidence justifies the routine administration of chemoprophylaxis to close contacts. We suggest that the appropriate response should be to routinely inform all household contacts of a patient with invasive group A streptococcal disease about the clinical manifestations of invasive disease and to seek immediate medical attention if they develop such symptoms.

Antimicrobial susceptibility testing of Streptococcus pneumoniae: quality assessment results.

Six strains of Streptococcus pneumoniae were distributed to 405 United Kingdom laboratories who were asked to test the susceptibility of the strains to penicillin, tetracycline, chloramphenicol and erythromycin and to provide details of methodology to test the standards of susceptibility testing. High error rates were seen only in failure to detect moderate resistance to penicillin (12%) and resistance to chloramphenicol (16%). Increased error rates were associated with several methods or practices. These included the use of certain culture media; failure to standardise the inoculum; inoculation by loop rather than by swab; failure to use control organisms; failure to measure zone sizes; the use of discs containing a high content of penicillin to test susceptibility to penicillin, and the use of high content discs for testing erythromycin, tetracycline, and chloramphenicol.

Pneumococci causing invasive disease in Britain 1982-1990.

A total of 5348 isolates of Streptococcus pneumoniae was serotyped and screened for insusceptibility to tetracycline, penicillin, erythromycin and chloramphenicol. Of these, 4238 (79%) were isolated from patients who had pneumonia or meningitis or were bacteraemic. Altogether, 3948 (74%) of the isolates belonged to one or other of the serotypes 1, 3, 4, 6, 8, 9, 14, 19 or 23 with serotypes 6, 14, 18, 19 and 23 being frequent causes of invasive disease in young children. Many isolates of type 1 were isolated from pneumonia and few from meningitis. Some 768 (14%) isolates were insusceptible to one or more antibiotic and 591 of these belonged to serotypes 6, 9, 14, 19 or 23. Representatives of type 14 resistant to erythromycin were prominent from 1986 onwards. There was an increase in the number of multi-resistant pneumococci from 1985. Among these were isolates of type 23 insusceptible to penicillin, chloramphenicol and tetracycline and cultures of type 6 resistant additionally to erythromycin.

Distribution and transferability of plasmids encoding trimethoprim resistance in urinary pathogens from Greece.

Of 505 strains of Enterobacteriaceae responsible for significant bacteriuria and isolated from hospital patients in two Greek cities in 1989, 151 strains (30%) were resistant to trimethoprim (MIC greater than or equal to 4 mg/L) and 220 (44%) were resistant to sulphamethoxazole (MIC greater than or equal to 64 mg/L); 127 (84%) of the trimethoprim-resistant strains exhibited high-level resistance (MIC greater than 1024 mg/L) and 121 (80%) were additionally resistant to four or more other antibiotics. Plasmids were detected in 141 (93%) of the trimethoprim-resistant strains. Trimethoprim resistance was encoded on self-transmissible plasmids in 79 (52%) of the resistant strains, and in a further seven strains (5%), plasmids coding for trimethoprim resistance could be mobilised by X+ factor. Co-transfer of various other antimicrobial resistances with trimethoprim resistance was observed, tetracycline resistance being the most common. The low degree of linkage observed between trimethoprim resistance and resistance to streptomycin and spectinomycin suggests that Tn7 is relatively uncommon in Greece. Classification of trimethoprim-resistance plasmids on the basis of their antimicrobial-resistance patterns and molecular mass revealed 39 different profiles. Overall, these findings differ from those from other European countries where the prevalence of transferable high-level trimethoprim resistance is low and where chromosomal Tn7-encoded trimethoprim resistance is common.

Virulence regulon polymorphism in group A streptococci revealed by long PCR and implications for epidemiological and evolutionary studies.

A method based on long PCR amplification and restriction endonuclease analysis of the virulence regulon was developed for a rapid (2 days), simple differentiation of group A streptococci. The PCR product size varied from 12.3 kb for serotypes M1 (NCTC 8198) and M12 (NCTC 10085) to 7.8 kb for serotype M6 (NCTC 8302). The fragment patterns formed on HaeIII digestion of the products were unique and this allowed the differentiation of each of the M-type strains (M1, M3, M4, M5, M6, M11, M12, M28, M76 and M78) studied. Contemporary M1 isolates all gave the same fragment pattern but differed from the prototype strain (NCTC 8198) in not having a 1.25-kb fragment. Isolates of serotypes M1 and M3 each had similar patterns, an indication of their clonality and global dispersion. In contrast, more than one restriction fragment length polymorphism (RFLP) pattern was detected among clinical isolates of serotypes M5, M6, M12, M4, M(R)28 and M78. Two strains that were M-protein non-typable by serological means were provisionally classified as M6 by comparisons of HaeIII long PCR fragment patterns.

An outbreak of serious illness and death among injecting drug users in England during 2000.

An outbreak of serious illness and death occurred in injecting drug users during 2000 in Scotland, Ireland and England. National and international collaboration was necessary for the investigation and management of this outbreak. In England and Wales active case-finding was initiated, coupled with standardised data collection and microbiological investigation of cases. Twenty-six definite or probable cases were identified in England between 1 April and 31 Aug. 2000; 17 of these occurred in the North. The overall case fatality was 50% (13/26). The principal apparent risk factor was a history of intramuscular or subcutaneous injection of heroin and the limited duration of the outbreak suggested that the problem might have been related to a particular supply of heroin. Clostridium novyi was isolated from two English cases. Taken in conjunction with contemporaneous microbiological and epidemiological results from Scottish and Irish cases, the probable aetiology for this outbreak was infection with C. novyi associated with both a particular supply of heroin and the method of preparation and injection used. A 'toolkit' was distributed in Sept. 2000 to all Consultants for Communicable Disease Control in England and Wales to assist them with the ongoing surveillance, investigation and management of this condition. Lessons learned have been used to produce guidance for the investigation and management of outbreaks of unexplained serious illness of possible infective aetiology.

Gentamicin resistance in clinical isolates of Escherichia coli encoded by genes of veterinary origin.

Seven (27%) of 26 gentamicin-resistant human clinical isolates of Escherichia coli were resistant to the veterinary aminoglycoside antibiotic apramycin. A gentamicin-resistant Klebsiella pneumoniae isolate from a patient infected with gentamicin/apramycin-resistant E. coli was also resistant to apramycin. DNA hybridisation studies showed that all gentamicin/apramycin-resistant isolates contained a gene encoding the enzyme 3-N-aminoglycoside acetyltransferase type IV (AAC[3]IV) that mediates resistance to gentamicin and apramycin in bacteria isolated from animals. Seven of the eight gentamicin/apramycin-resistant isolates were also resistant to the veterinary antihelminthic agent hygromycin B, a phenomenon observed previously in gentamicin/apramycin-resistant Enterobacteriaceae isolated from animals. Resistance to gentamicin/apramycin and hygromycin B was co-transferable in six of the isolates. Restriction enzyme analysis of plasmids in apramycin-resistant transconjugants derived from E. coli and K. pneumoniae isolates from the same patient were virtually identical, suggesting that inter-generic transfer of plasmids encoding apramycin resistance had occurred in vivo. These findings support the view that resistance to gentamicin and apramycin in clinical isolates of E. coli results from the spread of resistant organisms from animals to man, with subsequent inter-strain or inter-species spread, or both, of resistance genes on transferable plasmids.

Isolation and identification of Clostridium spp. from infections associated with the injection of drugs: experiences of a microbiological investigation team.

Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease. C. novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly. The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification. Additional phenotypic tests that are useful in recognising the main pathogenic species are described. Differentiation of C. novyi type A from C. botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture.

Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C. perfringens and Bacillus cereus isolated from injecting drug users during 2000.

As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

Distribution of serovariants of group B streptococci in isolates from England and Norway.

The distribution of capsular polysaccharide antigen (CHO) types, surface-exposed c proteins alpha (c alpha) and beta (c beta) and an R-protein antigen was examined in 334 group B streptococci (GBS) isolates from three groups of patients hospitalised in England and Wales or Norway. The isolates were from 108 carriers, 67 cases of neonatal infection and 154 cases of adult infection. Each group contained all CHO types (Ia, Ib, II, III, IV, V and NT); type III strains predominated except in the adult infected group. Strains within each CHO type could be further subdivided by the protein markers into five subtypes by a combined typing system. The proportion of type Ib and type III strains in the neonatal infection cases and of type Ib strains in the adult infection cases significantly outnumbered isolates of these serotypes among the carrier strains. Twenty-nine different serovariants were identified; 24, 13 and 23 serovariants among the carrier, neonatal infection and adult infection isolates, respectively. Certain CHO antigen-protein associations were identified, notably those between Ia/c alpha, Ib/c alpha beta and III/R. The proportion of invasive isolates that expressed protein was not higher than in the carrier isolates. All CHO-type Ib isolates contained a c protein, but 7% of the Ib isolates did not contain any of these proteins. These findings indicate that this combined typing approach may be useful in examining epidemiological problems associated with GBS.

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK.

OBJECTIVE: To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK. METHODS: During 1995-97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone. RESULTS: Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mefA gene was detected in seven isolates (five serotype 14 and two serotype 19F), ermB in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone. CONCLUSIONS: The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.

The emergence of vancomycin resistance in renal dialysis.

Intraperitoneal vancomycin is used in the treatment of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). We describe the emergence of low-level glycopeptide-resistance in five Gram-positive species over a one-year period. Isolation of these organisms was associated with vancomycin treatment failure in four patients who had had numerous episodes of peritonitis. Clinicians and microbiologists should be aware that repeated administration of glycopeptides to such patients might lead to the emergence of organisms resistant to these antibiotics.

Outbreak of infection in two UK hospitals caused by a strain of Klebsiella pneumoniae resistant to cefotaxime and ceftazidime.

During an 8-month period, Klebsiella pneumoniae resistant to cefotaxime and ceftazidime were isolated from 18 elderly patients in two closely-situated UK hospitals. Amongst these 18 patients, the organisms were isolated from urine samples of 17, from blood cultures of two and from a wound swab of one. The infected patients were located in nine different wards and several of the patients had been transferred between wards, within and between the two hospitals. All the bacterial isolates belonged to serotype K62, were non-typable or reacted only weakly with bacteriophage, showed similar plasmid profiles and were resistant to tetracycline and trimethoprim, thus indicating they were the same strain. Resistance to cefotaxime and ceftazidime was inhibited by clavulanic acid suggesting the involvement of extended-spectrum beta-lactamase (ESBL) enzyme activity. This was confirmed by analytical isoelectric focusing, which showed that isolates each produced two beta-lactamases with isoelectric points of 7.0(SHV-3) and 7.6 (SHV-1/2) respectively.

Nosocomial spread of Staphylococcus aureus showing intermediate resistance to methicillin.

A nosocomial outbreak of infection and colonization involving six patients and caused by a strain of Staphylococcus aureus showing intermediate resistance to methicillin (MIC = 4-8 mg l-1) is described. The outbreak was associated with skin-carriage of the epidemic strain by a nurse suffering from severe eczema. The reduced susceptibility of the outbreak strain to methicillin was associated with beta-lactamase production. Elimination or inhibition of beta-lactamase activity produced a two-fold decrease in methicillin MIC. There was no evidence for the presence of either penicillin-binding protein 2a or the corresponding mec gene, which mediate resistance in fully methicillin-resistant strains.