Environmental sampling coupled with real-time PCR and genotyping to investigate the source of a Q fever outbreak in a work setting.

A Q fever outbreak was declared in February 2016 in a company that manufactures hoists and chains and therefore with no apparent occupational-associated risk. Coxiella burnetii infection was diagnosed by serology in eight of the 29 workers of the company; seven of them had fever or flu-like signs and five had pneumonia, one requiring hospitalisation. A further case of C. burnetii pneumonia was diagnosed in a local resident. Real-time PCR (RTi-PCR) showed a widespread distribution of C. burnetii DNA in dust samples collected from the plant facilities, thus confirming the exposure of workers to the infection inside the factory. Epidemiological investigations identified a goat flock with high C. burnetii seroprevalence and active shedding which was owned and managed by one of the workers of the company as possible source of infection. Genotyping by multispacer sequence typing (MST) and a 10-loci single-nucleotide polymorphism (SNP) discrimination using RTi-PCR identified the same genotype (MST18 and SNP type 8, respectively) in the farm and the factory. These results confirmed the link between the goat farm and the outbreak and allowed the identification of the source of infection. The circumstances and possible vehicles for the bacteria entering the factory are discussed.

Variability of Bartonella genotypes among small mammals in Spain.

In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas-247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)-by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.

Short-term bactericidal activity of amoxicillin and cefotaxime against penicillin-susceptible and -resistant pneumococcal strains: an in vitro pharmacodynamic simulation.

The 8-hour in vitro activity of serum-simulated concentrations of amoxicillin (obtained after 875 mg oral dose) and cefotaxime (obtained after a 1 g i.v. dose), against 20 strains of the 5 Streptococcus pneumoniae serotypes most prevalent in Spain, was explored. Despite a greater initial inocula decrease observed with cefotaxime against the resistant strains at the first sampling time, a decrease > or =99.9% was obtained with both beta-lactams from 6h onwards against the penicillin-susceptible strains; the same was observed for the penicillin-resistant strains with amoxicillin but not with cefotaxime.

Streptococcus pneumoniae in children in Spain: 1990-1999.

UNLABELLED: This study analyses the serogroups/types (SGTs) and resistance to penicillin and erythromycin of 3921 strains isolated from 1990 to 1999 in children aged 0-14 y in Spanish hospitals of all the autonomous communities. Based on the age of the children, strains have been divided into five groups: 0-6 mo, > 6-1 y, > 1-2 y, > 2-5 y and > 5 y. While only eight SGTs were responsible for 80% of the infections in children from 6 mo to 2 y of age, this number increased to 11 and 16 for the groups > 2-5 y and > 5-14 y, respectively. SGTs 6, 14 and 19 were prevalent in blood and otic exudates. SGTs 1, 4, 5, 12 and 18 were more frequent in invasive disease but serotype 3 was clearly associated with otitis. Serotypes I and 5 were quite significant in children of over 2 y of age, and this should be taken into account in future vaccine formulations. CONCLUSION: Although high, the rate of penicillin resistance in the paediatric population has remained stable in recent years. Conversely, erythromycin resistance is still increasing in our country. Coverage by the 7-valent vaccine was 78 and 81% for blood and otic isolates, respectively. These coverage levels would be increased by 9% and 3% if 9-valent (plus 1 + 5 serotypes) were used and by an additional 2.6% and 7.6% using the 11-valent (plus 3 + 7) formulation.

A possible novel Francisella genomic species isolated from blood and urine of a patient with severe illness.

Two identical isolates were recovered in pure culture from the blood and urine of a patient suffering from severe septicaemia associated with obstructive pyelonephritis secondary to lithotripsy. Preliminary phenotypic and genotypic characterizations based on serological, biochemical and sequence analyses following PCR amplification of selected gene regions indicate that this organism represents a potential new Francisella genomic species.

Detection of Coxiella burnetii in ticks collected from Central Spain.

A total of 1482 adult ticks collected from vegetation and animals in central Spain in 2003-2005 were tested for the presence of Coxiella burnetii by polymerase chain reaction and subsequent reverse line blot hybridization (PCR-RLB). C. burnetii was identified in 7.7% of questing ticks (80/1039) and 3.4% of ticks collected from animals (15/443) belonging to four species: Hyalomma lusitanicum, Dermacentor marginatus, Rhiphicephalus sanguineus, and R. pusillus. These findings show an active role of ticks in maintaining C. burnetii in wild and peridomestic cycles in central Spain.

Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain.

The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.

Dot blot assay for the serotyping of pneumococci.

To simplify the serotyping of Streptococcus pneumoniae, a dot bot assay has been developed and compared with the standard quellung reaction in 1,082 isolates. The technique has been demonstrated to be sensitive, specific, easy to perform, and inexpensive. The dot blot assay could be useful when large numbers of pneumococci have to be studied.

Nucleotide sequence and chromosomal location of L-lactate dehydrogenase gene from Streptococcus pneumoniae.

We report here the 1244-bp sequence of a Streptococcus pneumoniae chromosomal fragment that contains the putative promoter and protein-coding region of the lactate dehydrogenase gene (ldh). The nucleotide sequence predicts a protein of 327 aa with a molecular weight of 35,202 daltons, after removal of the N-terminal methionine residue. The ldh gene is located on the ApaI fragment 1 and SmaI fragment 2 of the previously reported physical map of S. pneumoniae chromosome.

Identification of the psaA gene, coding for pneumococcal surface adhesin A, in viridans group streptococci other than Streptococcus pneumoniae.

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.

Beta-lactam modification of the bacteraemic profile and its relationship with mortality in a pneumococcal mouse sepsis model.

A sepsis BALB/c mice model was used to investigate the relationship between mortality and the bacteraemic profile produced by a serotype 6B Streptococcus pneumoniae clinical isolate (MIC/MBC of amoxicillin 4/4 mg/L and of cefotaxime 2/4 mg/L). Animals were treated subcutaneously with doses of amoxicillin or cefotaxime ranging from 6.25 to 50 mg/kg tds for 48 h, starting 1 h after intraperitoneal inoculation (2 x 10(7) cfu/mouse). Blood cultures were carried out daily over 15 days. A survival rate of 100% was obtained with amoxicillin 25 mg/kg and of 60% with cefotaxime 50 mg/kg. A statistically significant (P = 0.012) relationship was found between the maximum cfu/mL in blood and mortality. A maximum log cfu/mL of 6.5 was associated with an 84% probability of death.

Periodate oxidation of R36A pneumococci greatly enhances production of hybridomas secreting anti-protein antibodies.

Most hybridomas derived from mice immunized with non-capsulated Streptococcus pneumoniae strains secrete antibodies to C-polysaccharide epitopes and very rarely against cell wall proteins. Mild periodate oxidation of the non-capsulated R36A pneumococcal strain destroys C-polysaccharide antigenicity without a noticeable loss in the immunizing capacity for proteins. Following immunization of BALB/c mice with periodate-treated R36A cells, most hybridomas obtained (87.5%) secreted anti-pneumococcal protein antibodies. This strategy can be exploited for the development of MAb to pneumococcal polypeptides which, in turn, will be of use in the analysis of pneumococcal cell wall protein antigens.

Cloning, sequencing, and chromosomal location of a putative class-II aldolase gene from Streptococcus pneumoniae.

The nucleotide sequence of a 1620-bp chromosomal fragment from Streptococcus pneumoniae, containing a putative class-II aldolase gene, has been determined. The N-terminal amino acid (aa) sequence of S. pneumoniae class-II aldolase protein allowed us to determine the initiation site for the putative aldolase gene, and a molecular weight of 31,274 Da was predicted for the protein, after removal of the N-terminal methionine. Northern hybridization and primer extension analysis showed a 1100-nucleotide transcript with a transcription start site located 43 or 42 bp upstream of the start codon. Southern hybridization studies indicated that the putative class-II aldolase gene was in the ApaI fragment 6, SmaI fragment 9, and SacII fragment 12 or 13 of the physical map of S. pneumoniae chromosome. Southern hybridization analysis and partial sequencing performed in another eight streptococcus species, belonging to six different phylogenetic groups, suggested that a class-II aldolase gene with a considerable DNA homology to that of the S. pneumoniae, could exist in these streptococcal species.

Antimicrobial susceptibility and pneumococcal serotypes.

The increase in antibiotic resistance and the possible changes in serotype prevalence as a consequence of a new conjugated vaccine have contributed to renewed interest in the study of pneumococcal serotypes and their antibiotic resistances. Spain still has one of the highest penicillin resistance rates, but in the past 4-5 years a slight decrease has been observed. The level of resistance has not increased either, 12.7% of the 11 165 isolates studied showed high-level penicillin resistance but 94% of these had an MIC of only 2 mg/L. Serotypes 6, 9, 14, 19 and 23 included 83% of the penicillin-resistant pneumococci; the remaining 17% belonged to 18 different serotypes. We analysed these minor penicillin-resistant serotypes in view of their potential increase following a possible child vaccination programme. Four of these serotypes (11, 15, 21 and 35) were the most prevalent, and among them serotype 15 was particularly frequent with >50% of its strains resistant. The effective control of these minor penicillin-resistant serotypes should be based on continuous surveillance of pneumococcal epidemiology.

Beta-lactam activity against resistant pneumococcal strains is enhanced by the immune system.

Pandemic resistance in Streptococcus pneumoniae is compromising antibiotic activity. Antibiotics that act on the cell wall, such as beta-lactams, may have a combined effect with the immune system against S. pneumoniae, since both act on the bacterial envelope. This combined effect can be studied in vitro or in vivo with respect to bacterial killing, since lysis is the end-point of both beta-lactams and the immune system. We review here the in vitro increase in the bactericidal activity of aminopenicillins by non-specific immunity (complement and polymorphonuclear leucocytes). Few data are available on the collaboration of specific immunity and beta-lactams. We also review the effect of the presence of specific antibodies on the in vivo T > MIC needed for the therapeutic efficacy of amoxicillin, and on blood bacterial clearance in animal models. The effect that immunity has on pharmacodynamic parameters, such as T > MIC, in non-human studies may be used as a tool to predict the effect of these pharmacodynamic variations in overcoming resistance and its selection, in the context of increasing the use of pneumococcal conjugated vaccines.

Effects of specific antibodies against Streptococcus pneumoniae on pharmacodynamic parameters of beta-lactams in a mouse sepsis model.

A dose-ranging study to investigate the in vivo effects of the presence of specific antibodies on the efficacy of beta-lactam treatment of sepsis caused by Streptococcus pneumoniae (non-beta-lactam-susceptible serotype 6B isolate) was performed with a BALB/c mouse model. Hyperimmune serum was obtained from mice immunized with the heat-inactivated strain. The rate of mortality was 100% in nontreated animals in the absence of specific antibodies. A single injection of a one-half or one-quarter dilution of hyperimmune serum produced 60 to 40% survival rates. In the absence of specific antibodies, the minimal effective doses of amoxicillin and cefotaxime that produced survival rates of 100 and 80% were 25 and 50 mg/kg of body weight (three times a day for up to six doses), respectively. These doses produced times that the levels in serum remained above the MIC (deltaT > MICs) approximately 30% of the dosing interval. When specific antibodies were present (by administration of a one-half or one-quarter dilution of hyperimmune serum), the minimal effective doses of the antibiotics were 3.12 and 6.25 mg/kg ( approximately 8 times lower), with the deltaT > MICs being approximately 3 and 5% of the dosing interval for amoxicillin and cefotaxime, respectively. This in vivo combined pharmacodynamic effect offers possibilities that can be used to address penicillin resistance.

Modification of bacteraemia by specific antibodies and relation with mortality in a pneumococcal mouse sepsis model.

The relationship between mortality and the bacteraemic profile was investigated in a pneumococcal (serotype 6B) sepsis BALB/c mouse model where animals received protection by specific hyperimmune serum. A single intraperitoneal dose of hyperimmune serum obtained from mice immunized with the heat-inactivated strain was administered (non-diluted or diluted to 1/4 or to 1/16) to 5-mice study groups 1 h prior to intraperitoneal inoculation with the infective inoculum (3.57 x 108 cfu/ml). Blood cultures were performed daily over 15 days, with 8 microl of blood being collected from the tail vein; the samples were resuspended in Todd-Hewitt broth containing 10% trisodium citrate and plated onto blood agar for colony counting. Animals included in the control group received placebo (PBS). Mortality was 100% in control animals within the first 48 h. Hyperimmune serum decreased and delayed mortality in a dose-related trend, producing 100%, 80%, 60% and 40% survival rates at 72, 96, 144 and 360 h, with non-diluted serum. Bacteraemic profiles with maximum colony counts > or =5 x 107 cfu/ml in blood during the follow-up period were related to > or =65% probability of death, regardless of the serum dilution administered.