Ernesto Liebana

Replicon Typing of Plasmids Carrying CTX-M or CMY β-Lactamases Circulating among Salmonella and Escherichia coli Isolates

ABSTRACT Replicon typing of plasmids carrying blaCTX-M or blaCMY β-lactamase genes indicates a predominance of I1 and A/C replicons among blaCMY-carrying plasmids and five different plasmid scaffolds associated with the different types of blaCTX-M genes (I1, FII, HI2, K, and N). These results demonstrate the association of certain β-lactamase genes with specific plasmid backbones.

Mycobacterium tuberculosis subsp. caprae subsp. nov.: A taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain

Isolates from the Mycobacterium tuberculosis complex cultured from caprine pathological tissue samples were biochemically and genetically characterized. The isolates were negative for nitrate reduction and niacin accumulation, they weakly hydrolysed Tween 80, were sensitive to pyrazinamide (50 micrograms ml-1) and were resistant to 1 and 2 micrograms tiophene-2-carboxylic acid hydrazide ml-1 but not to 5 or 10 micrograms tiophene-2-carboxylic acid hydrazide ml-1. Sequencing of the pncA gene revealed a polymorphism characteristic of M. tuberculosis, whereas oxyR, katG and gyrA sequences were characteristic of Mycobacterium bovis. The fingerprinting patterns obtained with IS6110, direct repeats and polymorphic G+C-rich sequence-associated RFLP and direct variable repeat-spacer oligonucelotide typing (spoligotyping) segregated these isolates from the other members of the complex. The results of this testing, together with the repeated association of this micro-organism with goats, suggest that a new member of this taxonomic complex not matching any of the classical species had been identified. This unusual mycobacterium may play a role in the epidemiology of animal and human tuberculosis in Spain. The name Mycobacterium tuberculosis subsp. caprae subsp. nov. is proposed for these isolates. The type strain of Mycobacterium tuberculosis subsp. caprae subsp. nov. is gM-1T (= CIP 105776T).

blaCTX-M Genes in Clinical Salmonella Isolates Recovered from Humans in England and Wales from 1992 to 2003

ABSTRACT Cefotaximases (CTX-M) are a rapidly growing class A β-lactamase family that has been found among a wide range of clinical bacteria. One hundred and six isolates were selected from 278,308 Salmonella isolates based on resistance to ampicillin and cephalosporins and subjected to further characterization. Fourteen isolates were blaCTX-M PCR positive, and cefotaxime MICs for these isolates were ≥16 mg/liter. Furthermore, sequence analysis revealed the presence of type CTX-M9, -15, or -17 to -18. All 14 isolates presented different PFGE restriction profiles, although six Salmonella enterica serotype Virchow isolates formed a tight cluster. The blaCTX-M genetic determinants were present in transferable plasmids of ∼63, 105, and >148 kb. Plasmid restriction analysis showed that both horizontal transfer of similar plasmids among different clones and transfer of genes between different plasmids were likely mechanisms involved in the spread of blaCTX-M genes. We have found that CTX-M enzymes have emerged in community-acquired infections both linked to foreign travel and domestically acquired. This is the first report of a CTX-M enzyme in Salmonella in the United Kingdom. Also, it represents the first report of a blaCTX-M gene in Salmonella enterica serotype Stanley and a blaCTX-M-15 gene in Salmonella enterica serotypes Anatum, Enteritidis, and Typhimurium.

Comparison of gyrA mutations, cyclohexane resistance, and the presence of class I integrons in Salmonella enterica from farm animals in England and Wales.

ABSTRACT This study is focused on real-time detection of gyrA mutations and of the presence of class I integrons in a panel of 100 veterinary isolates of Salmonella enterica from farm animals. The isolates were selected on the basis of resistance to nalidixic acid, representing a variety of the most prevalent serotypes in England and Wales. In addition, organic solvent (cyclohexane) resistance in these isolates was investigated in an attempt to elucidate the presence of efflux pump mechanisms. The most prevalent mutation among the isolates studied was Asp87-Asn (n = 42), followed by Ser83-Phe (n = 38), Ser83-Tyr (n = 12), Asp87-Tyr (n = 4), and Asp87-Gly (n = 3). Two distinct subpopulations were identified, separated at the 1-mg/liter breakpoint for ciprofloxacin: 86% of isolates with mutations in codon 83 showed MICs of ≥1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of ≤0.5 mg/liter. Cyclohexane resistance was more prevalent among Ser83 mutants than among Asp87 mutants (34.7 and 4%, respectively), and in 79% of isolates that presented both gyrA mutations and cyclohexane resistance, the level of ciprofloxacin resistance was ≥2.0 mg/liter. Thirty-four isolates contained class I integrons, with 71% of the S. enterica serovar Typhimurium isolates and 6.9% of isolates belonging to other serotypes containing such elements. The methods used represent sensitive ways of investigating the presence of gyrA mutations and of detecting class-I integrons in Salmonella isolates. The results can be obtained in less than 1 h from single colonies without the need for purifying DNA.

Multiple Genetic Typing of Salmonella enterica Serotype Typhimurium Isolates of Different Phage Types (DT104, U302, DT204b, and DT49) from Animals and Humans in England, Wales, and Northern Ireland

ABSTRACT Salmonella enterica serotype Typhimurium is a common cause of salmonellosis among humans and animals in England, Wales, and Northern Ireland. Phage types DT104 and U302 were the most prevalent types in both livestock and humans in 2001. In addition, Salmonella serotype Typhimurium DT204b was responsible for a recent international outbreak involving England. A total of 119 isolates from humans (n = 28) and animals or their environment (n = 91), belonging to DT104 (n = 66), U302 (n = 33), DT204b (n = 12), and DT49 (n = 8), were fingerprinted by a combination of well-established genetic methods (pulsed-field gel electrophoresis [PFGE], PstI/SphI [PS] ribotyping, and plasmid profiling). The different techniques identified different degrees of polymorphism (from greatest to least, plasmid profiling [40 types], PS ribotyping [34 types], and PFGE [23 types]). It seems clear that a prevalent genomic clone, as well as a variety of less frequent clones, is present for each of the phage types. In most cases, the prevalent clones appeared within isolates from several animal species and from several geographical locations. We did not find clear evidence of a higher degree of diversity for any of the animal species included, or of any link between isolates from particular animal species and humans. The data presented show the inaccuracy of drawing epidemiological conclusions based on a single fingerprinting method. Strains that share one of the markers do not necessarily belong to the same clone, and a multiple typing approach is required to enable enough discrimination to track strains for epidemiological investigations.

Mycobacterium caprae Infection in Livestock and Wildlife, Spain

Mycobacterium caprae is a pathogen that can infect animals and humans. To better understand the epidemiology of M. caprae, we spoligotyped 791 animal isolates. Results suggest infection is widespread in Spain, affecting 6 domestic and wild animal species. The epidemiology is driven by infections in caprids, although the organism has emerged in cattle.

Restriction fragment length polymorphism and spacer oligonucleotide typing: A comparative analysis of fingerprinting strategies for Mycobacterium bovis

The combination of conventional investigation and DNA fingerprinting is yielding important insights into the epidemiology of Mycobacterium bovis infections. Various genetic markers used in restriction fragment length polymorphism (RFLP) have recently been exploited for fingerprinting of M. bovis isolates. The newly developed spacer oligonucleotide typing aimed to investigate the polymorphism of M. tuberculosis in the DR locus, has also been applied to the molecular typing of M. bovis isolates. This work compared the performance of the insertion sequence (IS) IS6110, IS1081 and the genetic elements polymorphic G + C-rich repeat (PGRS) and direct repeat (DR) used in RFLP analysis with spoligotyping using a group of 128 Spanish M. bovis isolates. In this study, the most sensitive technique for identifying polymorphism in M. bovis was PGRS-RFLP, closely followed by IS6110-RFLP. We propose several schemes for fingerprinting of these isolates, however, the clear geographical variations found by different authors makes the study of each local situation indispensable. An international consensus in the methods used would be desirable for efficient interlaboratory comparison of strains.

MYCOBACTERIUM GENAVENSE INFECTION IN CANARIES

A case of mycobacteriosis in a collection of canaries (Serinus canarius) is described. The affected birds showed nonspecific clinical signs and macroscopic lesions (slight splenomegaly). Histologic lesions found in liver, spleen, lungs, and kidneys consisted of noncaseous nodules containing accumulations of large macrophages that showed a highly vacuolated cytoplasm in which numerous acid-fast organisms were detected. Attempts to isolate and culture the organisms using Löwenstein-Jensen and Coletsos media proved unsuccessful. Microorganisms belonging to the species Mycobacterium genavense were identified by means of polymerase chain reaction techniques in hepatic tissue from both birds. This finding confirms the existence of fastidiously growing mycobacterial infections, other than Mycobacterium avium, in birds.

The insertion element IS6110 is a useful tool for DNA fingerprinting of Mycobacterium bovis isolates from cattle and goats in Spain

A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M.bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.

Use of polymerase chain reaction in the diagnosis of tuberculosis in cats and dogs

Samples from four dogs and four cats suspected of having tuberculosis were processed for histopathology, bacterial culture and polymerase chain reaction (PCR). A simple, rapid method for the extraction of DNA from tissue samples was used in two PCR assays designed to confirm the diagnosis of tuberculosis. The PCR assays detected all the culture-positive samples from these animals and no false positive results were obtained. The PCR technique was successful for the direct detection of organisms from the Mycobacterium tuberculosis complex and reduced the time needed for a diagnosis to two days.