Allelic polymorphism of human FcγRIIA-H/R131 receptor in American tegumentary leishmaniasis

FcγRIIA binding to IgG subclasses with different levels of affinity is influenced by the polymorphism in the gene that encodes this receptor. The substitution of arginine (R) for histidine (H) in the 131 position defines three allelic patterns, H/H, R/R, and H/R, resulting in FcγRIIA-H/H131 affinity for IgG2 and higher affinity for IgG3 subclasses. Studies have shown the importance of genetic host factors in leishmaniasis and participation of FcγRs on the macrophage infection by amastigote forms and in the immune response to Leishmania sp. We analysed the influence of allelic diversity patterns of the receptor FcγRIIA on American tegumentary leishmaniasis (ATL). FcγRIIA-H/R131 polymorphism was determined by PCR followed by an allele-specific enzymatic digestion in 88 individuals with ATL and 98 healthy volunteer blood donors (control group). The genotypic and allelic distributions of FcγRIIA-H/R131 were similar among the studied groups as well in mild and severe clinical forms of ATL. Our results suggest no association between this allelic polymorphism and susceptibility or resistance to ATL, neither influencing the development of different clinical forms of this illness.     

Antigenic specificity of the 72-kilodalton major surface glycoprotein of Leishmania braziliensis braziliensis.

We examined the expression and the antigenicity of the major surface polypeptides of Leishmania braziliensis braziliensis and Leishmania donovani chagasi, parasites which commonly coexist in the same endemic areas of Bolivia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles from surface-iodinated promastigotes showed the presence of a unique iodinatable polypeptide of 72 kDa on the L. b. braziliensis surface and of two major components of 65 and 50 kDa exposed at the surface of L. d. chagasi. Comparison of the peptide digestion profiles of the major iodinated polypeptides of both strains showed no similarity between the maps of the 72- and the 65-kDa polypeptides of L. b. braziliensis and L. d. chagasi, respectively. Immunoprecipitation of surface-labeled L. b. braziliensis Nonidet P-40 extracts with 35 serum specimens obtained from Bolivian patients with cutaneous and mucocutaneous leishmaniasis showed that all serum specimens recognized predominantly the 72-kDa antigen and high-molecular-mass proteins in some cases. The recognition patterns were independent of the geographical origin of the patient, the type of lesion, and the serum antibody titer. Serum specimens from children with visceral leishmaniasis did not precipitate the L. b. braziliensis 72-kDa antigen. Hamster hyperimmune serum against L. b. braziliensis also recognized the 72-kDa surface antigen. However, this recognition was inhibited in the presence of the homologous nonlabeled antigen but not in the presence of heterologous (L. d. chagasi and Trypanosoma cruzi) antigens. The specific recognition of 72-kDa surface antigen in both natural and experimental L. b. braziliensis infections suggests that this antigen could be a good candidate for use in the differential immunodiagnosis and prognosis of the disease.     

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting.

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.     

Antigenicity of Leishmania braziliensis histone H1 during cutaneous leishmaniasis: localization of antigenic determinants

The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that L. braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L. braziliensis histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant L. braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.     

DNA sequencing confirms the involvement of Leishmania (L.) amazonensis in American tegumentary leishmaniasis in the state of São Paulo, Brazil

INTRODUCTION: American tegumentary leishmaniasis (ATL) represents one of the most important public health issues in the world. An increased number of autochthonous cases of ATL in the Northeastern region of S?o Paulo State has been documented in the last few years, leading to a desire to determine the Leishmania species implicated. METHODS: PCR followed by DNA sequencing was carried out to identify a 120bp fragment from the universal kDNA minicircle of the genus Leishmania in 61 skin or mucosal biopsies from patients with ATL. RESULTS: DNA sequencing permitted the identification of a particular 15bp fragment (5' GTC TTT GGG GCA AGT... 3') in all samples. Analysis by the neighbor-joining method showed the occurrence of two distinct groups related to the genus Viannia (V) and Leishmania (L), each with two subgroups. Autochthonous cases with identity to a special Leishmania sequence not referenced in Genbank predominated in subgroup V.1, suggesting the possible existence of a subtype or mutation of Leishmania Viannia in this region. In the subgroup L.2, which showed identity with a known sequence of L. (L.) amazonensis, there was a balanced distribution of autochthonous and non-autochthonous cases, including the mucosal and mucocutaneous forms in four patients. The last observation may direct us to new concepts, since the mucosal compromising has commonly been attributed to L. (V.) braziliensis, even though L. (L.) amazonensis is more frequent in the Amazonian region. CONCLUSIONS: These results confirm the pattern of distribution and possible mutations of these species, as well as the change in the clinical form presentation of ATL in the S?o Paulo State.     

In vitro responses of human peripheral blood mononuclear cells to whole-cell, particulate and soluble extracts of Leishmania promastigotes

Whole-cell and soluble extracts of Leishmania promastigotes have both been used as skin test antigens and have also been tested as vaccine candidates. However, the differences in antigenicity between soluble and particulate Leishmania fractions are not known. We evaluated in vitro responses of PBMC from 30 American tegumentary leishmaniasis (ATL) patients and seven noninfected donors to different antigen preparations from Leishmania promastigotes, namely Leishmania amazonensis and L. braziliensis whole-cell extracts, as well as soluble and particulate fractions of L. amazonensis. All Leishmania antigen preparations stimulated significantly higher proliferation and interferon (IFN)-gamma production (but not interleukin (IL)-10 production) in PBMC from the leishmaniasis patients than in cells from the control subjects. The L. braziliensis whole-cell extract stimulated significantly higher cell proliferation and IFN-gamma production than the L. amazonensis whole-cell extract in the group of patients but not in the control group. This result can be explained by the fact that the patients were infected with L. braziliensis. Again in the group of patients, the PBMC proliferative responses as well as the levels of IFN-gamma and IL-10 stimulated by L. amazonensis whole-cell extract were significantly greater than those elicited by the L. amazonensis soluble fraction but were not significantly different from those elicited by the L. amazonensis particulate fraction. We found a higher antigenicity of the particulate fraction as compared to the soluble fraction, what suggests that the antigens present in the particulate fraction account for most of the antigenicity of whole-cell Leishmania promastigote antigen extracts.     

PCR typing of Corynebacterium diphtheriae by random amplification of polymorphic DNA.

The usefulness of random amplification of polymorphic DNA (RAPD) analysis with Ready-To-Go RAPD beads was investigated for the rapid differentiation of Corynebacterium diphtheriae isolates from Eastern Europe and neighbouring countries. A selection of 45 C. diphtheriae isolates of known origin, biotype, toxigenicity status and ribotype were examined by RAPD. Twenty RAPD profiles (designated Rp1-Rp20) were revealed among the 45 isolates. There was 100% correlation between RAPD profiles and ribotypes. Preliminary studies showed that the use of crude DNA preparations resulted in poor amplification and the patterns were not reproducible. Different thermal cycler models produced different RAPD profiles from the same DNA sample. Reproducibility of the technique was good when the same thermal cycler was used throughout. RAPD proved to be a simple and a rapid method for analysing C. diphtheriae and it is a method which can be used as a potential alternative to ribotyping or as a screening technique during outbreak investigations.     

Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania

Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.     

Epidemiology of American tegumentary leishmaniasis in domestic dogs in an endemic zone of western Venezuela

Domestic dogs are not only reservoir hosts of the American zoonotic visceral leishmaniasis (ZVL) but of the American zoonotic tegumentary leishmaniasis (ATL) as well, for different reasons. However it is still controversial to state that dogs are incriminated as ATL reservoir hosts as there is evidence that humans and dogs are likely to be exposed in the same way to sandfly vector. In Venezuela this issue has not been completely addressed, for this reason we selected a location inside Trujillo city to study eco-epidemiological conditions as well as to survey a significant sample of dogs by Montenegro Skin Test (MST). Antigen was prepared according to standard procedure using Leishmania (V) braziliensis promastigotes (80 microg/ml); response was read 48 hours post-inoculation with an induration size > 5 mm being considered as positive. The study place is an endemic mountainous semi-urban area located at 850-950 masl with an average rainfall of 150 mm/year. We evaluated 61 dogs in 46 houses with 168 human beings. Among the human population 27 cases of ATL were reported (16.1%). With the MST we found 19 positive-reaction dogs (31%) (mean MST size of 9.58 mm, 95% CI: 8.41-10.75) in 13 houses (28%). Multivariate analysis did not reveal significant association between domestic MST positive-dog ownership and human ATL cases (RR = 1.48, p = 0.28). Although some studies have indicated that dog ownership and dog infection rates are associated with an increased risk of human disease in different evaluated places, this question has not been completely answered in Venezuelan studied zones, further research is necessary.     

Identification and differentiation of Trichophyton rubrum clinical isolates using PCR-RFLP and RAPD methods

Trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. Several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. The purpose of this study was to apply selected DNA fingerprinting methods to the identification and strain discrimination of T. rubrum clinical isolates. Fifty-seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and strain differentiation using a randomly amplified polymorphic DNA (RAPD) method, with two primers designated 1 and 6. Using PCR-RFLP, 55 of the isolates studied were confirmed to be T. rubrum. Among those, a total of 40 and five distinct profiles were obtained by RAPD with primers 1 and 6, respectively. The combination of profiles from both RAPD assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. A dendrogram analysis performed on the profiles generated by RAPD with primer 1 showed most of the isolates (87.3%) to be genetically related. PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum species, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing.     

Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil

Numerical zymotaxonomy and variability of the internal transcribed spacers (ITS) between the small and large subunits of the rRNA genes were used to examine strain variation and relationships in natural populations of Leishmania (Viannia) braziliensis. A total of 101 strains from distinct hosts and Brazilian geographic regions were assigned to 15 zymodemes clustered in two major genetic groups. The great number of isolates (48.5%) placed in zymodeme IOC/Z-27 were collected on the Atlantic coast. The high molecular diversity found in populations in the Amazon Basin was related to the great number of sandfly vector(s) in that region. The results of the restriction fragment length polymorphism analysis of the ITS depicted considerable intraspecific variation. Genotypic groups A, B, and C contained 39, 40, and 22 isolates, which were divided into 16, 10, and 15 genotypes, respectively. The genetic polymorphism observed demonstrates the degree of diversity of L. (V.) braziliensis strains from different regions where they are endemic. The results reinforce the clonal theory for Leishmania parasites showing the genetic diversity of this pathogen and an association of L. (V.) braziliensis genotypes with specific transmission cycles, probably reflecting an adaptation of different clones to the vector species involved.     

Random amplified polymorphic DNA profiles of Trypanosoma cruzi isolates from chagasic patients with different clinical forms

The genetic variability of 61 Trypanosoma cruzi isolates from 47 chronic chagasic patients of Minas Gerais state was analyzed by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) using M13-40, λgt11-F, and L15996 primers. Cluster analysis by unweighted pair group method analysis was applied to RAPD profiles, and cluster analysis used to verify a possible correlation among different clinical forms of the disease from these patients. The T. cruzi isolates showed distinct grouping on tree topology, with the isolates not being possible to establish a correlation to the clinical forms of Chagas’ disease. These data showed that the T. cruzi isolates from these patients would compose a group of populations well correlated genetically.     

Upgrading the flow-cytometric analysis of anti-Leishmania immunoglobulins for the diagnosis of American tegumentary leishmaniasis

We have previously described a flow cytometry-based assay to detect anti-live Leishmania (Viania) braziliensis promastigote antibodies (FC-ALPA) with prominent performance of FC-ALPA to diagnosis American tegumentary leishmaniasis (ATL). However, the laboriousness to work with live parasites represented the major drawback for using FC-ALPA in routine clinical laboratory. Herein, we have presented an upgraded technology using fixed Leishmania (Leishmania) amazonensis promastigotes as antigen (FC-AFPA). Our data demonstrated that FC-AFPA-IgG displays outstanding performance for ATL diagnosis with high sensitivity (99%) and specificity (100%). Moreover, Likelihood Ratio indicated that positive results (LR+) has an infinite times more chance to come from ATL than from non-infected individuals (NI). Despite the high frequency of cross-reactivity with putative ATL co-endemic diseases, including visceral leishmaniasis, Chagas disease and malaria, FC-AFPA-IgG showed remarkable potential for differential diagnosis with other dermatological illnesses such as leprosy and sporotrichosis. FC-AFPA-IgG subclasses analysis revealed that LTA is characterized by IgG1>IgG3>IgG2 = IgG4 anti-L. amazonensis profiling, electing FC-AFPA-IgG1 and IgG3 with better performances to diagnosis ATL diagnosis. Additionally, FC-AFPA-IgG3 showed to be a better diagnostic tool in endemic areas for malarial disease. Despite the substantial advance to work with fixed promastigotes that contributes to its higher sensitivity, the lower specificity of FC-AFPA represented the major flaws as compared to FC-ALPA, suggesting that further improvement is still required to minimize the cross-reactivity with trypanosomatidae infections. Perspectives for using a flow cytometry multiplex based methodology to simultaneously assess anti-L. amazonensis, anti-L. chagasi and anti-Trypanosoma cruzi IgG reactivity is currently under investigation.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay.

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Random amplified polymorphic DNA and phenotyping analysis of Salmonella enterica serovar enteritidis isolates collected from humans and poultry in Uruguay from 1995 to 2002

Molecular and phenotyping techniques were applied to study Salmonella enterica serovar Enteritidis strains both from human cases of infection and of avian origin isolated in Uruguay from 1995 to 2002. A group of 62 isolates was subjected to random amplified polymorphic DNA (RAPD) assay and analysis of antibiotic resistance patterns. Twenty-one of these strains were further characterized by phage typing and analysis of their protein expression profiles. RAPD fingerprinting with five different primers discriminated 10 different genetic profiles. Of the 62 strains tested, 48 had a single major genetic profile, whereas the other nine profiles were evenly distributed among the other strains. The genetic diversity was greater among strains of animal origin than among isolates of human origin. Comparative examination of the results obtained by RAPD analysis and phenotypic analysis and by strain source provided evidence of the reliable discriminatory power of RAPD analysis in our study. Six avian isolates with antibiotic resistance were detected: two were nalidixic acid resistant and four had a particular beta-lactam resistance pattern. The last four isolates all had the same unusual phage type (phage type 4b); however, RAPD analysis differentiated them into two groups. Two isolates with unique RAPD profiles were recovered from distinct human cases, suggesting that the technique differentiates unrelated strains. Overall, the results show the existence of a predominant genetic type that is present in poultry and that is transmitted to humans. There are also several other genotypes, but only a few of them could be recovered from human sources, suggesting the existence of different pathogenic traits among strains circulating in the country.     

Attempt to differentiate Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis using the SSR-PCR technique

The ability to differentiate reference strains of Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis was evaluated using the simple sequence repeat polymerase chain reaction (SSR-PCR) technique. This technique differentiates the Leishmania species, generating distinct DNA amplicon profiles. The SSR-PCR profiles were similar to but more reproducible than those produced by RAPD. SSR-PCR is presented as an alternative to other molecular methods for the differentiation of Leishmania species or strains.     

Genetic variability within the species Leishmania infantum by RAPD. A lack of correlation with zymodeme structure

The infraspecific variability of the species Leishmania infantum is studied by using genetic markers generated by random amplified polymorphic DNA (RAPD). We have applied this technique, using 18 primers of arbitrary sequence, to 33 strains of the parasite belonging to 18 zymodemes isolated in different clinical forms and hosts. Other strains belonging to the species L. donovani, L. major, L. tropica and L. mexicana were used as a reference. The RAPD technique produced very different genetic profiles between L. infantum and L. major, L. tropica and L. mexicana with all primers used, whereas 11 of the 18 primers distinguished L. infantum strains from the species L. donovani. All primers except 1 (TAF 300), generated polymorphism in the L. infantum strains. The dendrograms constructed with the isoenzyme data and with RAPD are congruent in relation to the separation of the different species but show little agreement within the L. infantum species, reflecting the genetic heterogeneity of the strains belonging to one zymodeme. A geographical structuralisation is observed with two diverging groups that evolve independently whereas there is no relation between the genotype of the parasite and the host or between the former and the clinical form of the disease.     

Proteome analysis of Leishmania (Viannia) braziliensis by two-dimensional gel electrophoresis and mass spectrometry

Leishmania (Viannia) braziliensis, a protozoan parasite widespread in the New World, is responsible for the infection of different mammal orders, including humans. This species is considered to be a major etiological agent of American cutaneous leishmaniasis. A proteomic study was carried out to identify proteins expressed by L. (V.) braziliensis. One hundred and one spots representing 75 protein entries were identified by MALDI-TOF-TOF. Isoelectric point values estimated by gel electrophoresis matched closely with predicted values, although some discrepancies existed suggesting that post-translational protein modifications may be common in L. braziliensis. Moreover, 20 hypothetical proteins were experimentally identified. Identified proteins were classified into 15 groups according to biological process. Among the proteins identified, approximately 40% have not been previously reported in a proteomic map of Leishmania. In addition, a number of potential virulence factors and drug targets were identified in this protein map, including some proteins associated with the metastatic phenotype. This study describes the first compilation of a proteomic reference map for L. braziliensis (pI 4-7, M(r) 10-130 kDa) and provides a very useful tool for comparative studies of strains isolated from patients presenting different clinical manifestations of leishmaniasis as well as a potential tool to identify markers for clinical diagnosis, therapeutics, and prognosis.     

Outbreak of Acinetobacter baumannii producing the carbapenem-hydrolyzing oxacillinase OXA-58 in Rome, Italy

In this study 45 epidemic and sporadic isolates of Acinetobacter baumannii were investigated by antimicrobial resistance, integron identifications and genotyping. Isolates were genotyped by random amplified polymorphism (RAPD) DNA and pulsed-field gel electrophoresis (PFGE). Four different RAPD patterns were observed among the isolates of our collection, further discerned in six PFGE types. Two prevalent genotypes were identified, one corresponding to a carbapenem resistant epidemic clone, causing an outbreak at the intensive care unit of a hospital of Rome. Two class 1 integrons, carrying different gene cassette arrays, were identified among the two prevalent genotypes. Nucleotide analysis of the integron-variable regions revealed the presence of the aacA4, orfO, bla(OXA-20), and aacC1, orfX, orfX', aadA1 gene cassette arrays, respectively. All the carbapenem resistant strains analyzed in this study carried the bla (OXA-58) gene located on plasmids.