Aránzazu Portillo

Macrolide Resistance Genes in Enterococcus spp.

ABSTRACT Seventy-eight isolates of different Enterococcusspecies (E. faecalis, n = 27; E. faecium, n = 23; E. durans,n = 8; E. avium, n = 6;E. hirae, n = 9; E. gallinarum, n = 3; and E. casseliflavus, n = 2) with a variety of erythromycin resistance phenotypes were examined for the presence of macrolide resistance genes (ermA, ermB,ermC, ermTR, mefA/E, andmsrA). Positive PCR amplifications of ermB were obtained for 39 of 40 highly erythromycin-resistantEnterococcus isolates (MICs, >128 μg/ml) of different species; the remaining highly resistant E. faecium isolate was positive for PCR amplification of ermA but was negative for PCR amplification of the ermB and ermCgenes. For all enterococcal strains for which erythromycin MICs were ≤32 μg/ml PCRs were negative for erm methylase genes. For all E. faecium isolates PCR amplified products of the expected size of 400 bp were obtained when msrA primers were used, with the results being independent of the erythromycin resistance phenotype. All the other enterococcal species gave negative results by msrA PCRs. Sequencing of the msrAPCR products from either erythromycin-susceptible, low-level-resistant, or highly resistant E. faecium strains showed that the amplicons did not correspond to the msrA gene described forStaphylococcus epidermidis but corresponded to a new putative efflux determinant, which showed 62% identity with themsrA gene at the DNA level and 72% similarity at the amino acid level. This new gene was named msrC.

Crimean-Congo Hemorrhagic Fever Virus in Ticks, Southwestern Europe, 2010

To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV; family Bunyaviridae, genus Nairovirus) causes outbreaks of severe hemorrhagic fever in humans, with case-fatality rates <30% (1,2). The disease was initially recognized by Russian scientists in the 1940s (3), and the virus was first isolated in the Democratic Republic of Congo some years later (4). CCHFV is reported throughout broad regions of Africa, Europe, the Middle East, and Asia. Reports linking transmission of the virus with an infected vector have involved ticks of the genus Hyalomma (5). It appears that maintenance of active foci of CCHFV in the field is dependent on Hyalomma spp., even within periods of silent activity. Several vertebrates are involved in the natural transmission cycle (6). Transmission of CCHFV to humans occurs through tick bites, direct contact with blood or tissues of infected animals, person-to-person spread, or by nosocomial infection (1). In southeastern Europe, the Balkans are the known western limit for CCHFV (7). This finding is of special interest because Hyalomma marginatum, the main tick vector in the western Paleartic (an ecozone that includes temperate and cold areas of Eurasia and North Africa and several archipelagos and islands in the Atlantic and Pacific Oceans), is common throughout the Mediterranean Basin (7), where clinical cases of the disease or the virus have not been reported. Unsupported claims of the effects of climate on virus distribution have been reported but never empirically demonstrated (8). We report the detection of CCHFV in ticks collected in southwestern Europe. A total of 117 semi-engorged adult H. lusitanicum ticks were collected from 28 adult red deer (Cervus elaphus) in November 2010, at a site (39.63°N, 7.33°W) in Caceres, Spain. Live ticks were transported to the special pathogens laboratory at Hospital San Pedro–CIBIR in Logrono (northern Spain), classified, and frozen at −80°C. For RNA extraction, specimens were washed in 70% ethanol and then in Milli-Q water (Milli-Q Advantage water system; Millipore Iberica, S.A., Madrid, Spain) that had been autoclaved. Each tick was cut lengthwise; half was used for additional processing and the remainder was stored. Before use, each half was crushed in sterile conditions. RNA was individually extracted by using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and frozen at −80°C. The RNA was distributed in 12 pools and retrotranscribed by using the Omniscript RT kit (QIAGEN) according to the manufacturer’s instructions and then frozen at −20°C. Nested PCRs were performed by using specific primers for the small segment of CCHFV as described (9). Negative controls (with template DNA but without primers and with primers and containing water instead of template DNA) were included in all assays. For the second round of PCRs, 2 of 12 pools showed amplicons of the expected size (211 bp). Only 1 amplicon could be sequenced. MEGA5 (www.megasoftware.net) was used to compare the sequence with representative small segment sequences of CCHFV available in GenBank (Figure). (Aligned sequences are available from the authors.) Pools of cDNA were submitted to the Spanish National Center of Microbiology (Madrid), where results were confirmed. The CCHFV sequence we report showed 98% genetic similarity (204/209 bp) with sequences recorded for CCHFV in Mauritania and Senegal, on the western coast of Africa. Figure Evolutionary relationships of Crimean-Congo hemorrhagic fever virus strains from Spain and other representative sites. Evolutionary history was inferred by using the unweighted pair group method with arithmetic mean. The optimal tree is shown (sum of ... This finding suggests the circulation of CCHFV in southwestern Europe. The close affinity of the strain from Spain with strains circulating in western Africa and the lack of similarity with isolates from eastern Europe suggest the introduction of this virus from nearby countries of northern Africa. Migratory movements of birds could explain the presence of the virus in southwestern Europe because birds are common hosts of immature H. marginatum, which was reportedly introduced into Europe through annual migratory flights along the western coast of Africa (10). Because of the lack of genetic similarities among virus strains, trade movements of domestic or wild ungulates from eastern Europe do not support our finding. We cannot state whether this virus was circulating previously or if other strains are present in the area because CCHFV detection in the western Mediterranean region has not been previously addressed. H. lusitanicum ticks exist as relatively isolated populations in a narrow strip from Sicily to Portugal. The Mediterranean rabbit and ungulates, the main hosts for immature and adult H. lusitanicum ticks, respectively, are residents of the collection area; however, the movement of these animals through trade has not occurred for several years. Thus, H. lusitanicum ticks could not serve as a spreading vector in the western Mediterranean region. The CCHFV strain from southwestern Europe has been found in ticks restricted to hosts that cannot spread long distances. Therefore, although it would be unlikely, given the strain’s similarity with CCHFV isolates from Senegal and Mauritania, we should not exclude the possibility of an ancient existence for this strain. Additional data collected in the Mediterranean Basin are necessary to establish the actual range of CCHFV.

A patient from Argentina infected with Rickettsia massiliae.

The first confirmed case of Rickettsia massiliae infection in the New World (Buenos Aires, Argentina) is described. To date, only two cases of human infection had been reported in Europe. The patient, a woman, had a fever, a palpable purpuric rash on the upper and lower extremities, and a skin lesion (eschar) on the right leg compatible with tache noire. When interviewed, she reported having had contact with dog ticks. After treatment with doxycycline for 12 days, her symptoms resolved. Rickettsia massiliae infection was diagnosed by molecular-based detection of the microorganism in a biopsy specimen of the eschar.

Role of Birds in Dispersal of Etiologic Agents of Tick-borne Zoonoses, Spain, 2009

We amplified gene sequences from Anaplasma phagocytophilum, Borrelia garinii, B. valaisiana, B. turdi, Rickettsia monacensis, R. helvetica, R. sibirica sibirica, and Rickettsia spp. (including Candidatus Rickettsia vini) in ticks removed from birds in Spain. The findings support the role of passerine birds as possible dispersers of these tick-borne pathogens.

Crimean-Congo Hemorrhagic Fever Virus in Ticks from Migratory Birds, Morocco

Crimean-Congo hemorrhagic fever virus was detected in ticks removed from migratory birds in Morocco. This finding demonstrates the circulation of this virus in northwestern Africa and supports the hypothesis that the virus can be introduced into Europe by infected ticks transported from Africa by migratory birds.

Rickettsia sp. Strain Colombianensi (Rickettsiales: Rickettsiaceae): A New Proposed Rickettsia Detected in Amblyomma dissimile (Acari: Ixodidae) From Iguanas and Free-Living Larvae Ticks From Vegetation

ABSTRACT From January to December 2009, 55 Amblyomma dissimile (Koch) ticks removed from iguanas in the municipality of Monteria and 3,114 ticks [458 Amblyomma sp. larvae, 2,636 Rhipicephalus microplus (Canestrini) larvae and 20 Amblyomma sp. nymphs] collected over vegetation in Los Cordobas were included in the study. The ticks were pooled into groups from which DNA was extracted. For initial screening of Rickettsia sp., each pool was analyzed by gltA real-time polymerase chain reaction (PCR). Positive pools were further studied using gltA, ompA, and ompB conventional PCR assays. Sequencing and phylogenetic analysis were also conducted. Rickettsial DNA was found in 28 pools of ticks (16 A. dissimile pools and 12 free-living larvae pools) out of 113 (24.7%) using real-time PCR. The same 28 pools were also positive using conventional PCR assays aimed to amplify gltA, ompA, and ompB. For each gene analyzed, PCR products obtained from 4/28 pools (two pools of A. dissimile, one pool of Amblyomma sp. larvae and one pool of Rh. microplus larvae) were randomly chosen and sequenced twice. Nucleotide sequences generated were identical to each other for each of the rickettsial genes gltA, ompA, and ompB, and showed 99.4, 95.6, and 96.4% identity with those of Rickettsia tamurae. They were deposited in the GenBank database under accession numbers JF905456, JF905458, and JF905457, respectively. In conclusion, we present the first molecular evidence of a novel Rickettsia (Rickettsia sp. strain Colombianensi) infecting A. dissimile ticks collected from iguanas, and also Rh. microplus and unspeciated Amblyomma larvae from vegetation in Colombia.

Human Rickettsia sibirica mongolitimonae infection, Spain.

Human Rickettsia sibirica mongolitimonae Infection, Spain

A Confirmed Case of Rickettsia parkeri Infection in a Traveler from Uruguay

The first confirmed case of Rickettsia parkeri infection in Uruguay is reported. To date, in South America, molecularly confirmed cases of human infection have been found in Argentina and probably, Brazil. Our patient returned to Spain after a 7-day trip to Colonia Suiza (Southwestern Uruguay). He presented fever (39°C), chills, and two eschars (tache noire-like) surrounded by an indurated, erythematous halo on the inner side of the left ankle besides a maculopapular rash on the legs. After treatment with doxycycline for 7 days, he fully recovered. R. parkeri infection was diagnosed by molecular-based detection of the microorganism in a swab specimen of the eschar. Diagnosis was supported by seroconversion between acute- and convalescent-phase sera specimens.

Rickettsia Species in Ticks Removed from Humans in Istanbul, Turkey

A total of 167 ticks collected from humans in Istanbul (Turkey) in 2006 were screened for Rickettsia species, and nested PCRs targeting gltA and ompA rickettsial fragment genes were carried out. Rickettsia monacensis (51), R. aeschlimannii (8), R. conorii subsp. conorii (3), R. helvetica (2), R. raoultii (1), R. africae (1), R. felis (1), and other Rickettsia spp. (2), were detected. To our knowledge, these Rickettsia species (except R. conorii) had never been reported in ticks removed from humans in Turkey. The presence of R. africae also had not been previously described, either in Hyalomma ticks or in any European tick species. In addition, R. aeschlimannii and R. felis had not been found associated with Rhipicephalus bursa specimens. The presence of human pathogenic Rickettsia in ticks removed from humans provides information about the risk of tick-borne rickettsioses in Turkey.

Anaplasma spp. in Wild Mammals and Ixodes ricinus from the North of Spain

Our objectives were to investigate the presence of Anaplasma spp. infection in red deer, wild boars, and Ixodes ricinus removed from deer surveyed in La Rioja, as well as to analyze the presence of Anaplasma spp. in I. ricinus from different Spanish regions--ours included. A total of 21 deer and 13 wild boar blood samples as well as 295 I. ricinus removed from deer, vegetation, or asymptomatic people were tested by polymerase chain reaction targeting Anaplasma spp. 16S rRNA gene and groESL heat shock operon. Twelve deer blood samples were found to be infected with Anaplasma centrale (n = 7) or Anaplasma phagocytophilum (n = 5). No wild boar blood samples gave positive polymerase chain reaction results. Further, A. phagocytophilum was detected in 12 out of 89 I. ricinus removed from deer and in 18 out of 168 I. ricinus collected over vegetation in the North of Spain. Anaplasma spp. was not detected in any of the 38 I. ricinus removed from people. Nucleotide sequences for 16S rRNA gene showed substancial heterogeneity. The etiological agent of human anaplasmosis was found in two deer blood samples, an adult tick from deer, and a nymph from vegetation. The 16S rRNA sequences for 12 out of 35 samples matched the sequence of the Ap-variant 1 strain previously described in the United States, and the remaining 19 positive samples (deer blood and I. ricinus) showed variations with unknown significance. Although the groEL DNA sequences varied among analyzed strains, the deduced amino acid sequences did not change for any of them. This study suggests that deer population from La Rioja harbors strains of A. phagocytophilum similar to that pathogen for humans and other of unknown pathogenicity. Further, it seems that the Ap-variant 1 is circulating among I. ricinus ticks from the North of Spain more frequently than the A. phagocytophilum strain associated to human anaplasmosis.