Genomic Characterization of Staphylococcus aureus in Wildlife.

Staphylococcus aureus is an opportunistic multi-host pathogen that threatens both human and animal health. Animals can act as a reservoir of S. aureus for humans, but very little is known about wild animals' epidemiological role. Therefore, in this study, we performed a genomic characterization of S. aureus isolates from wildlife, hunters, and their auxiliary hunting animals of Eastern Spain. Of 20 different species, 242 wild animals were examined, of which 28.1% were S. aureus carriers. The common genet, the Iberian ibex, and the European hedgehog were the species with the highest S. aureus carriage. We identified 30 different sequence types (STs), including lineages associated with wild animals such as ST49 and ST581, multispecies lineages such as ST130, ST398, and ST425, and lineages commonly isolated from humans, including ST1 and ST5. The hunters and the single positive ferret shared ST5, ST398, or ST425 with wild animals. In wildlife isolates, the highest resistance levels were found for penicillin (32.8%). For virulence factors, 26.2% of them carried superantigens, while 14.8% harbored the immune evasion cluster (IEC), which indicates probable human origin. Our findings suggest that wild animals are a reservoir of clinically relevant genes and lineages that could have the potential to be transmitted to humans. These data support the notion that wildlife surveillance is necessary to better understand the epidemiology of S. aureus as a pathogen that circulates among humans, animals, and the environment.

Effect of selection for growth rate on the rabbit (Oryctolagus cuniculus) immune system and its response after experimental Staphylococcus aureus infection.

The aim of the work was to evaluate if genetic selection for daily gain may affect the immune system. Two experiments were performed. The first one involved 80 rabbit females and their first two litters to explore the effect of selection on the ability of animals to maintain immune competence. Two generations from a line selected for average daily gain (ADG) were evaluated (VR19 generation 19th, n = 43; VR37 generation 37th, n = 37). In females, the effect of selection and its interaction with physiological state were not significant for any trait. In litters, the selection criterion increased the granulocyte to lymphocyte ratio. The second experiment involved 73 19-week-old females (VR19, n = 39; VR37, n = 34) to explore the effect of genetic selection on immune response after S. aureus infection. The VR37 rabbit females had lower counts for total lymphocytes, CD5+, CD4+, CD8+, CD25+, monocytes, the CD4+/CD8+ ratio and platelets than those of VR19 (-14, -21, -25, -15, -33, -18, -11 and -11%, respectively; P < 0.05). VR37 had less erythema (-8.4 percentage points; P < 0.05), fewer nodules (-6.5 percentage points; P < 0.05) and a smaller nodule size (-0.65 cm3 on 7 day post-inoculation; P < 0.05) compared to VR19. Our study suggests that genetic selection for average daily gain does not negatively affect the maintenance of a competent immune system or the ability to establish immune response. It seems that such selection may improve the response to S. aureus infections.

Marked Presence of Methicillin-Resistant Staphylococcus aureus in Wild Lagomorphs in Valencia, Spain.

The appearance of methicillin-resistant strains of Staphylococcus aureus (MRSA) in several animal species (including rabbits) has set off alarms for their capacity to act as reservoirs for this bacterium. This is especially important in wild animals given its epidemiological implications. The objectives of this study were to identify and characterize S. aureus, specifically MRSA, strains in wild lagomorph high-density areas. Ten hares and 353 wild rabbits from 14 towns with a high rabbit density in the Valencian region (eastern Spanish coast) were sampled. Swabs from the nasal cavity, ears, perineum and lesions (when present) were taken for microbiological studies. The detection of different genes and antibiotic susceptibility studies were also carried out. Of all the animals, 41.3% were positive for S. aureus, of which 63.3% were MRSA. Ears were the anatomical location with more S. aureus and MRSA strains. The more frequently identified MLST type was ST1945 (97.1%, 136/140). The mecA gene was found only in one sample. The rest (n = 139) carried the mecC gene and were included in CC130, except one. Penicillin resistance was detected in 28 mec-negative isolates and, in one case, bacitracin resistance. mecA isolate presented resistance to enrofloxacin and tetracycline, and 10 mecC isolates also showed bacitracin resistance. No MRSA isolate was positive for genes chp, sea, tst and PVL. Two ST1945 isolates contained IEC type E (comprising genes scn and sak). mecA-isolate was positive for blaZ. Of the 28 MSSA strains showing resistance to penicillin, 22 carried the blaZ gene. These surprising results highlight the marked presence of MRSA strains in wild rabbits in high-density areas.

Pathogenesis of Intradermal Staphylococcal Infections: Rabbit Experimental Approach to Natural Staphylococcus aureus Skin Infections.

Despite the enormous efforts made to achieve effective tools that fight against Staphylococcus aureus, the results have not been successful. This failure may be due to the absence of truly representative experimental models. To overcome this deficiency, the present work describes and immunologically characterizes the infection for 28 days, in an experimental low-dose (300 colony-forming units) intradermal model of infection in rabbits, which reproduces the characteristic staphylococcal abscess. Surprisingly, when mutant strains in the genes involved in virulence (JΔagr, JΔcoaΔvwb, JΔhla, and JΔpsmα) were inoculated, no strong effect on the severity of lesions was observed, unlike other models that use high doses of bacteria. The inoculation of a human rabbitized (FdltBr) strain demonstrated its capacity to generate a similar inflammatory response to a wild-type rabbit strain and, therefore, validated this model for conducting these experimental studies with human strains. To conclude, this model proved reproducible and may be an option of choice to check both wild-type and mutant strains of different origins.

Differences in virulence between the two more prevalent Staphylococcus aureus clonal complexes in rabbitries (CC121 and CC96) using an experimental model of mammary gland infection.

Staphylococcal mastitis is a major health problem in humans and livestock that leads to economic loss running in millions. This process is currently one of the main reasons for culling adult rabbit does. Surprisingly, the two most prevalent S. aureus lineages isolated from non-differentiable natural clinical mastitis in rabbits (ST121 and ST96) generate different immune responses. This study aimed to genetically compare both types of strains to search for possible dissimilarities to explain differences in immune response, and to check whether they showed similar virulence in in vitro tests as in experimental intramammary in vivo infection. The main differences were observed in the enterotoxin gene cluster (egc) and the immune-evasion-cluster (IEC) genes. While isolate ST121 harboured all six egc cluster members (seg, sei, selm, seln, selo, selu), isolate ST96 lacked the egc cluster. Strain ST96 carried a phage integrase Sa3 (Sa3int), compatible with a phage integrated into the hlb gene (ß-haemolysin-converting bacteriophages) with IEC type F, while isolate ST121 lacked IEC genes and the hlb gene was intact. Moreover, the in vitro tests confirmed a different virulence capacity between strains as ST121 showed greater cytotoxicity for erythrocytes, polymorphonuclear leukocytes and macrophages than strain ST96. Differences were also found 7 days after experimental intramammary infection with 100 colony-forming units. The animals inoculated with strain ST121 developed more severe gross and histological mastitis, higher counts of macrophages in tissue and of all the cell populations in peripheral blood, and a significantly larger total number of bacteria than those infected by strain ST96.

Characterization of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates Obtained From Commercial Rabbitries Located in the Iberian Peninsula.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have been a growing problem in human medicine since the 1960s, and more recently in veterinary medicine with the appearance of livestock-associated MRSA (LA-MRSA). Nevertheless, information about the presence of MRSA in rabbits is quite scarce since only one LA-MRSA identification has been previously reported. The present study aimed to determine genotypic characterization by verifying the presence of resistance determinants, virulence, and toxin genes of different S. aureus strains that cause lesions in rabbits, and their phenotypic traits based on the antimicrobial susceptibility profile. The analysis of 240 S. aureus isolates obtained from different lesion types collected from 89 Spanish and Portuguese rabbit commercial farms in the last 4 years (2014-2017) was performed. The methicillin-resistant gene mecA was found in 11.25% of the studied isolates (27 of 240) from 19 farms (13 Spanish and 6 Portuguese). Staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed type III (n = 15). Additionally, three MRSA isolates carrying the mecC gen were detected in samples from three different farms (two Spanish and one Portuguese). None of the 30 MRSA isolates was PVL-positive or tst-positive. After the multilocus sequence typing (MLST) procedure, 16 belonged to ST2855, 6 to ST146, 6 to ST398, and 2 ST4774. No ST121 isolate was mec-positive. ST398 and ST4774 isolates lacked the immune-evasion-cluster (IEC) genes. ST2855 strains were associated with the presence only of the sak gene, and ST146 isolates were ascribed to IEC type E. Therefore, this is the first description of LA-MRSA from rabbits belonging to ST2855. Interestingly, one ST2855 and two ST4774 isolates were mecC-positive, which could act as a mecC-MRSA reservoir. More studies are needed to further characterize these isolates and their relationship with humans and other animal species.

Vaccine Failures in Patients Properly Vaccinated with 13-Valent Pneumococcal Conjugate Vaccine in Catalonia, a Region with Low Vaccination Coverage.

Vaccine failures occurring with 13-valent pneumococcal conjugate vaccine (PCV13) in 3 pediatric hospitals in Barcelona (2012-2013) are described. PCV13 vaccine failure was defined as the occurrence of an invasive pneumococcal infection in children properly vaccinated by PCV13. Among 84 patients with invasive pneumococcal infection, 32 had received at least one dose of PCV13. Seventeen of them had invasive pneumococcal infection produced by a PCV13 serotype. Among those, 9 patients were considered to have a PCV13 vaccine failure. Serotype 3 was isolated in 6 patients, serotype 19A in 2 and serotype 6B in 1.

Host responses associated with chronic staphylococcal mastitis in rabbits.

Staphylococcal infection causes substantial economic losses in commercial rabbit production systems, and is associated with a wide variety of lesions, including chronic suppurative mastitis, which mainly affects breeding females. Most chronic staphylococcal infections in rabbits are caused by the ST121 lineage of Staphylococcus aureus, although other less common lineages, such as ST96 can also be involved. The aims of the present study were to characterise the host immune response in natural cases of mastitis in rabbits caused by S. aureus, to evaluate any relationship between peripheral and local immunity and to investigate the effect of different S. aureus genotypes on these immune responses. Adult multiparous female rabbits that were affected with chronic staphylococcal mastitis (n = 204) were enrolled into the study. Histological and immunohistochemical evaluations of mammary glands were undertaken, as well as flow cytometric analyses of blood. S. aureus isolates from the mammary glands were identified by multilocus sequence typing. Differences in the number of infiltrating cells were detected, depending on the type of pathology, with more immature lesions demonstrating greater cellularity, characterised by greater numbers of T lymphocytes, macrophages and plasma cells. A relationship was seen between the cells in blood and mammary tissues, the most notable being the positive correlation between monocytes and tissue macrophages. When glands were infected with ST96 strains, fewer granulocytes (P < 0.01) and greater numbers of B cells (P < 0.01), T cells (P < 0.001), CD4(+) T cells (P < 0.001) and CD8(+) T cells (P < 0.01) were detected, compared with mammary glands that were infected by ST121 strains of S. aureus.

A single natural nucleotide mutation alters bacterial pathogen host tropism.

The capacity of microbial pathogens to alter their host tropism leading to epidemics in distinct host species populations is a global public and veterinary health concern. To investigate the molecular basis of a bacterial host-switching event in a tractable host species, we traced the evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it evolved through a likely human-to-rabbit host jump over 40 years ago and that only a single naturally occurring nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one that could infect rabbits. Related mutations were identified at the same locus in other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report of a single mutation that was sufficient to alter the host tropism of a microorganism during its evolution highlights the capacity of some pathogens to readily expand into new host species populations.

Performance of a rapid multi-analyte 2-photon excitation assay in children with acute respiratory infection.

The purpose of this study is to evaluate the diagnostic performance of the novel 2-photon excitation-based mariPOC© Assay (ArcDia Laboratories, Turku, Finland) for antigen detection of respiratory viruses versus real-time polymerase chain reaction (PCR). The mariPOC Assay and 2 multiplex real-time PCR techniques were performed on nasopharyngeal samples from pediatric patients with suspicion of acute respiratory infection admitted to a children's hospital in Spain during October 2011 to January 2013. A total of 233 samples were studied. Sensitivities and specificities (95% confidence interval) of the mariPOC Assay were for respiratory syncytial virus (RSV), 78.4% (69.7-85.6) and 99.2% (96.3-100.0); influenza virus (IFV) A, 66.7% (26.2-94.0) and 99.6% (97.9-100.0); IFV-B, 63.6% (33.6-87.2) and 100.0% (98.7-100.0); human metapneumovirus (hMPV), 60.0% (34.5-81.9) and 100.0% (98.6-100.0); adenovirus (ADV), 12.5% (0.6-48.0) and 100.0% (98.7-100.0), respectively. The mariPOC Assay is a highly specific method for simultaneous detection of 8 respiratory viruses but has sensitivities that range from moderately high for RSV to moderate for IFV and hMPV and low for ADV.

Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries.

BACKGROUND: Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. METHODS: Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. RESULTS: Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). CONCLUSION: Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.

Are risk factors associated with invasive pneumococcal disease according to different serotypes?

The aim of this study was to investigate risk factors for the most common serotypes of invasive pneumococcal disease (IPD). A total of 293 IPD cases were analyzed in children aged 3-59 mo in a community with intermediate vaccination coverage with the 7-valent pneumococcal vaccine (PCV7). IPD cases were reviewed during 2007-2009 in two pediatric hospitals in Catalonia (Spain). A multivariate analysis using unconditional logistic regression was performed to estimate the adjusted odds ratio. PCV7 coverage was 45.4%. Pneumonia with empyema (64.5%) was the most frequent clinical manifestation. The most common serotypes were: serotype 1 (21.2%), 19A (16.0%), 3 (12.6%) and 7F/A (6.8%). 70.0% of serotypes found were included in the 13-valent conjugate vaccine (PCV13), 39.2% in the 10-valent conjugate vaccine and 8.1% in the PCV7. PCV7 was protective in IPD cases due to PCV7-serotypes (aOR: 0.15, 95% CI:0.04-0.55). Serotype 1 was positively associated with attending day care or school (aOR: 3.55, 95% CI: 1.21-10.38) and age 24-59 mo (aOR: 7.70, 95% CI:2.70-21.98). Serotype 19A was positively associated with respiratory infection in the previous month (aOR: 2.26, 95% CI: 1.03-4.94), non-penicillin susceptible IPD (aOR: 1.89, 95% CI:1.13-3.16) and negatively associated with age 24-59 mo (aOR: 0.19, 95% CI:0.09-0.41). Serotype 3 was positively associated with vaccination (aOR: 4.87, 95% CI:2.05-11.59). No factors were associated with serotype 7F/A. Vaccination with pneumococcal vaccines including more serotypes may reduce the risk of disease in our setting.

Risk factors for invasive pneumococcal disease in a community with a high proportion of non vaccine serotypes.

The aim of this study was to investigate factors associated with vaccination with 7-pneumococcal conjugate vaccine (PCV7) and risk factors for invasive pneumococcal disease (IPD) and for penicillin-nonsusceptible strains in a community with intermediate vaccination coverage. We conducted a prospective, matched case-control study in children aged 3-59 months with IPD admitted to two hospitals in Catalonia. Three controls matched by hospital, age, sex, date of hospitalization and risk medical conditions were selected for each case. We calculated odds ratios for potential risk factors using logistic regression. Of the 1075 children included, 46.6% were considered fully vaccinated by age. 91.1% of cases were caused by non-PCV7 serotypes. Vaccination with PCV7 was positively associated with attending day care or school and negatively associated with age 24-59 months, >4 cohabitants and low social class. Attending day care or school and >4 cohabitants were risk factors for IPD. Previous antibiotic treatment in children aged 24-59 months was a protective factor for IPD; however, antibiotic use in the previous month and age <24 months were associated with penicillin-nonsusceptible IPD. In a community where IPD in children aged <5 years is caused mainly by non-PCV7 Streptococcus pneumoniae serotypes and where vaccine coverage is only intermediate, attending day care or school, age <24 months, >4 cohabitants and social class were associated with vaccination. Attending day care or school was a strong risk factor for IPD, while vaccination was protective in children aged <24 months. Age and antibiotic use in the previous month were associated with penicillin-nonsusceptible IPD.

Prevalence and clonal distribution of pcpA, psrP and Pilus-1 among pediatric isolates of Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of vaccine-preventable deaths globally. The objective of this study was to determine the distribution and clonal type variability of three potential vaccine antigens: Pneumococcal serine-rich repeat protein (PsrP), Pilus-1, and Pneumococcal choline binding protein A (PcpA) among pneumococcal isolates from children with invasive pneumococcal disease and healthy nasopharyngeal carriers. We studied by Real-Time PCR a total of 458 invasive pneumococcal isolates and 89 nasopharyngeal pneumococcal isolates among children (total = 547 strains) collected in Barcelona, Spain, from January 2004 to July 2010. pcpA, psrP and pilus-1 were detected in 92.8%, 51.7% and 14.4% of invasive isolates and in 92.1%, 48.3% and 18% of carrier isolates, respectively. Within individual serotypes the prevalence of psrP and pilus-1 was highly dependent on the clonal type. pcpA was highly prevalent in all strains with the exception of those belonging to serotype 3 (33.3% in serotype 3 isolates vs. 95.1% in other serotypes; P<.001). psrP was significantly more frequent in those serotypes that are less apt to be detected in carriage than in disease; 58.7% vs. 39.1% P<.001. Antibiotic resistance was associated with the presence of pilus-1 and showed a negative correlation with psrP. These results indicate that PcpA, and subsequently Psrp and Pilus-1 together might be good candidates to be used in a next-generation of multivalent pneumococcal protein vaccine.

Rapid and easy identification of capsular serotypes of Streptococcus pneumoniae by use of fragment analysis by automated fluorescence-based capillary electrophoresis.

The purpose of this study was to develop a high-throughput method for the identification of pneumococcal capsular types. Multiplex PCR combined with fragment analysis and automated fluorescent capillary electrophoresis (FAF-mPCR) was utilized. FAF-mPCR was composed of only 3 PCRs for the specific detection of serotypes 1, 2, 3, 4, 5, 6A/6B, 6C, 7F/7A, 7C/(7B/40), 8, 9V/9A, 9N/9L, 10A, 10F/(10C/33C), 11A/11D/11F, 12F/(12A/44/46), 13, 14, 15A/15F, 15B/15C, 16F, 17F, 18/(18A/18B/18C/18F), 19A, 19F, 20, 21, 22F/22A, 23A, 23B, 23F, 24/(24A/24B/24F), 31, 33F/(33A/37), 34, 35A/(35C/42), 35B, 35F/47F, 38/25F, and 39. In order to evaluate the assay, all invasive pneumococcal isolates (n = 394) characterized at Hospital Sant Joan de Déu, Barcelona, Spain, from July 2010 to July 2011 were included in this study. The Wallace coefficient was used to evaluate the overall agreement between two typing methods (Quellung reaction versus FAF-mPCR). A high concordance with Quellung was found: 97.2% (383/394) of samples. The Wallace coefficient was 0.981 (range, 0.965 to 0.997). Only 11 results were discordant with the Quellung reaction. However, latex reaction and Quellung results of the second reference laboratory agreed with FAF-mPCR for 9 of these 11 strains (82%). Therefore, we considered that only 2 of 394 strains (0.5%) were not properly characterized by the new assay. The automation of the process allowed the typing of 30 isolates in a few hours with a lower cost than that of the Quellung reaction. These results indicate that FAF-mPCR is a good method to determine the capsular serotype of Streptococcus pneumoniae.

Viral load at diagnosis and influenza A H1N1 (2009) disease severity in children.

To assess viral load at diagnosis (VLAD) as a biomarker of novel influenza disease severity, epidemiologic and clinical data of admitted patients <18 years old with Influenza A H1N1 (2009) infection and respiratory symptoms were prospectively collected in a single pediatric tertiary hospital, from weeks 30-51 of 2009. Seventy patients were included. VLAD in children who had symptoms for ≥ 5 days was an accurate parameter distinguishing the patients who required mechanical ventilation (MV) from those who did not required it (area under the ROC curve: 0.73; P=0.03). Having <4.5 log10 copies/ml with ≥ 5 days of symptoms was associated with a lower risk of requiring MV.

Viral coinfection in children less than five years old with invasive pneumococcal disease.

Seventy-one patients <5 years of age who were hospitalized with invasive pneumococcal disease were studied in the period between August 2008 and December 2009. The purpose was to determine the proportion of episodes that were coinfected with respiratory virus. Viral coinfection was common (44/71; 62%), with rhinovirus and influenza virus being the most frequently detected. Highly invasive serotypes (1, 5, 7F, 14, 19A) were found in 31 of 71 patients, of whom 15 had viral coinfection (15/31; 48%). Viral detection occurred significantly more often in those episodes caused by nonhighly invasive serotypes (29/40; 72%), suggesting that a viral synergism could help those serotypes to make invasiveness more likely.