Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli strains isolated from two early stages of poultry production.

Our aim was to monitor the resistance of Campylobacter isolates from two initial stages of broiler production in 5 grandparent breeder broiler farms (GPBFs) and 12 parent breeder broiler farms (PBFs) in which no antimicrobials were used during the study. Susceptibility tests were carried out for 805 strains (697 Campylobacter jejuni and 108 Campylobacter coli) against nalidixic acid, ciprofloxacin, erythromycin, amoxicillin, amoxicillin plus clavulanic acid, tetracycline, gentamicin, and chloramphenicol using the disk-diffusion method. Quinolone resistance was the most abundant overall (74.9%) and at each stage of production. The second largest resistance was for tetracycline with 48.2%. The resistance against amoxicillin plus clavulanic acid, gentamicin, and chloramphenicol was not found. The percentages of resistance and multidrug-resistant (MDR) isolates were always higher in the PBFs than in the GPBFs. However, pan-susceptible populations (total 10.3%) were isolated in our survey. C. coli isolates were more resistant to tetracycline and erythromycin (96.3% and 23.1%, respectively) than for C. jejuni (40.7% and 0%, respectively) and were more MDR (33.3% vs. 11.9%). In conclusion, as other authors have shown, even in the absence of antibiotic pressure, relatively high rates of quinolone resistance are found in Campylobacter. However, a decrease in quinolone resistance has been observed compared to other studies in Spain [i.e., 99%; Saenz et al. Antimicrob. Agents Chemother. 2000;44(2):267-271]. MDR, fluoroquinolone-, macrolide-, and tetracycline-resistant Campylobacter populations are issues of concern in public health.

Characterization of multidrug-resistant Enterobacteriaceae carrying plasmid-mediated quinolone resistance mechanisms in Spain.

OBJECTIVES: The aim of this study was to detect and characterize plasmid-mediated quinolone resistance determinants as well as genes responsible for additional resistances in Enterobacteriaceae isolates in Spain. METHODS: The resistance genes were identified by PCR and sequencing. Plasmid analysis was carried out by S1-PFGE and PCR-based replicon typing. Conjugation assays were performed to link resistance genes to plasmids. The genetic relationships among the strains were determined by XbaI-PFGE. RESULTS: One hundred and twenty-three isolates carried qnr as the only quinolone resistance determinant. One Salmonella Bredeney was positive for qnrB2 harboured on a 320 kb conjugative IncHI2 plasmid. One Salmonella Newport was positive for qnrB4 harboured on a 70 kb conjugative IncFIIs plasmid. Twenty-five Salmonella Thompson were positive for qnrA1. Twenty-two harboured a 220 kb non-conjugative and non-typeable plasmid, two a 220 kb conjugative IncHI2 plasmid and one a 120 kb non-conjugative IncA/C plasmid. qnrS1 was always detected on non-conjugative ColE(TP) plasmids of various sizes. Thus, two Salmonella Montevideo strains carried a 20 kb plasmid while Salmonella Typhimurium strains carried plasmids of 10 kb (n = 91) or 30 kb (n = 2). One Escherichia coli was positive for qnrA1 detected on a 220 kb conjugative IncHI2 plasmid. qnr alleles and ß-lactamases were associated in Salmonella Bredeney (harbouring bla(SHV-12)), Salmonella Newport (harbouring bla(DHA-1)) and E. coli (harbouring bla(CTX-M-9)). CONCLUSIONS: This is the first epidemiological study of qnr genes in Enterobacteriaceae isolates from Spain. Salmonella plasmids bearing qnr alleles are not a localized phenomenon in Spain and wide variation in plasmids and co-resistance was detected. The presence of qnr determinants in Salmonella serotypes commonly reported in human disease is concerning.

Drinking water as the source of Campylobacter coli infection in grandparent heavy breeders.

The aim of the present study was the molecular identification of a common source of infection of Campylobacter coli in two grandparent breeder farms. Campylobacter jejuni and C. coli were isolated from well water and cloacal swabs from grandparent chickens. Colonies were genotyped using restriction fragment length polymorphism-flaA gene, pulsed field gel electrophoresis and multi-locus sequence typing. The same genotype of C. coli was found in both farms and in the well from which drinking water was supplied to the farms. The well water was epidemiologically linked as the source of C. coli infection. The molecular identification for epidemiological source-tracking of C. coli in breeder farms could aid in combating the colonization of this pathogen and therefore to reduce their incidence in human campylobacteriosis.

Emergence of extended-spectrum beta-lactamases and AmpC-type beta-lactamases in human Salmonella isolated in Spain from 2001 to 2005.

OBJECTIVES: To study the resistance to third-generation cephalosporins in Salmonella strains isolated from humans in a 5 year period in Spain, and to identify the responsible genes and their dissemination. METHODS: Twenty-seven isolates were analysed by PCR and sequencing to identify the genes responsible for the beta-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli K12J53, plasmid detection with S1-PFGE, hybridization and PCRs of the transconjugants. The genetic relationship was determined by PFGE. RESULTS: We found bla(CTX-M-9) and bla(CTX-M-10) in Salmonella Virchow PT19. bla(CTX-M-14) was detected in Salmonella (IV) 44:z(4),z(23):-, Salmonella Enteritidis PT6a, Salmonella Typhimurium DT193 and Salmonella Typhimurium DT104B. bla(CTX-M-1) was found in Salmonella Litchfield. bla(CTX-M-15) and bla(CTX-M-32) were found in Salmonella Enteritidis PT1. bla(SHV-12) was found in Salmonella Blockley, Salmonella Hadar PT2, Salmonella Enteritidis PT21, Salmonella Enteritidis PT1 and Salmonella Bredeney. bla(SHV-2) was found in Salmonella Livingstone. bla(CMY-2) was detected in Salmonella Bredeney, Salmonella Newport, Salmonella Enteritidis PT5b and Salmonella Heidelberg. bla(DHA-1) was detected for the first time in Spain in Salmonella Newport. One strain of Salmonella Senftenberg harboured two extended-spectrum beta-lactamases, bla(SHV-12) and bla(CTX-M-9). We have found a large variety of beta-lactamase families as well as several members of major relevance, such as CTX-M-15, CTX-M-32, CMY-2 and DHA-1. XbaI-PFGE, conjugation assays and S1-PFGE hybridization showed that all these beta-lactamases were mediated by plasmids. CONCLUSIONS: This study demonstrates the emergence of a public health risk related to resistance to beta-lactams in Salmonella. The resistance trends need to be monitored carefully.

Salmonella enterica serotype Typhimurium carrying hybrid virulence-resistance plasmids (pUO-StVR): a new multidrug-resistant group endemic in Spain.

The epidemiological impact in Spain of an emerging group of multidrug-resistant Salmonella enterica serotype Typhimurium, characterized by the presence of virulence-resistance hybrid plasmids (termed pUO-StVR) that are related to the S. Typhimurium virulence plasmid pSLT, was evaluated. Adscription to the group was based on detection of the bla(OXA-1) gene (encoding ampicillin resistance) by PCR, and identification of a pUO-StVR plasmid through hybridization with specific probes for virulence (spvC) and resistance (bla(OXA-1)) genes. In this way, 57 out of 134 ampicillin-resistant clinical isolates of S. Typhimurium, collected over 2002-2004 in 21 Spanish cities, were assigned to the group, which can be already regarded as endemic. Most isolates (>89%) shared the following features: (i) resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides, and tetracycline, encoded by bla(OXA-1)-catA1-aadA1-sul1-tet(B); (ii) a class 1 integron (InH) with the bla(OXA-1)-aadA1 gene cassettes within its variable region of ca. 2000bp; (iii) the spvC, rck, samA, oriT, traT, traX, repA (RepFIIA), and parA/B genes (but not rsk and pefABCD) of pSLT; (iv) a hybrid plasmid of ca. 125kb, termed pUO-StVR2, where the resistance and virulence genes are located. However, intra-group diversity was also detected, since a total of four resistance phenotypes, five resistance genotypes, two integron profiles, five plasmid variants (pUO-StVR2, 4-7, differing in size, restriction profile and/or resistance pattern), 15 XbaI-BlnI combined macrorestriction profiles, and five phage types were identified. Each hybrid plasmid was revealed as a distinctive BlnI band, through hybridization with pUO-StVR2. The genetic markers used, together with the knowledge generated in the present study, could be applied to epidemiological surveillance of S. Typhimurium pUO-StVR worldwide.

Identificación de una cepa de Vibrio cholerae O1como responsable de casos de enfermedaddiarréica aguda de sucesivos brotes de cólera en Guinea Ecuatorial / No disponible

Fundamento: De 2004 a 2006 se produjeron varios brotes de Enfermedad Diarreica Aguda (EDA) en Guinea Ecuatorial. La etiología de los brotes se llevó a cabo mediante una investigación microbiológica. Material y Métodos: Se realizaron coprocultivos de los enfermos con EDA. Las colonias sospechosas se identificaron bioquímicamente para establecer su especie. Las cepas de Vibrio choleraese serotipificaron por aglutinación con antisueros específicos de serogrupo y serotipo y se determinó la resistencia frente a un grupo de antimicrobianos. Se investigó por PCR el gen ctxA de la toxinacolérica y el gen responsable de la resistencia al vibriostático O129. La tipificación molecular se llevó a cabo por PFGE con la enzima NotI. Resultados: Las cepas se identificaron como V.cholerae O1, serotipo Inaba, resistente al vibriostáticoO129 y a los antimicrobianos estreptomicina, trimetoprim y sulfametoxazol. Se amplificó el genctxA y se determinó la presencia del gen dfrA1 responsable de la resistencia cruzada O129-trimetoprim.Todas fueron indistinguibles cuando se analizaron por PFGE. Conclusiones: Los brotes de EDA en Guinea Ecuatorial tuvieron como causa una cepa de V.cholerae O1, serotipo Inaba, con el mismo perfil de resistencias a antimicrobianos e idéntico pulsotipo, lo que sugería el origen clonal de las cepas estudiadas (AU)

Basis: From 2004 to 2006 several outbreaks of Acute Diarrhoeal Diseases (ADD) happened in Guinea Equatorial. The etiological agent of outbreaks was determined by means of microbiological investigation. Materials and Methods: Stool samples from patients were cultured. For specie identification, biochemical examination of suspicious colonies was performed. Serotyping of Vibrio Cholerae strains was carried on by slide agglutination. The sensitivity to different antimicrobians was determined. Amplification of the ctx A gene and the O129 resistance gene was carried out by PCR assays. Molecular typing of isolates was performed using PFGE with the restriction enzyme NotI. Results: All tested strains were identified as V.cholerae O1, serotype Inaba, resistant to the vibriostaticO129 and to the antibiotics streptomycin, trimethoprim and sulfamethoxazole. The ctxA gene and the dfrA1 gene responsible of the O129-trimethoprim cross resistance, were PCR amplified. All PFGEpatterns were identical. Conclusions: The consecutive Guinea Equatorial ADD outbreaks arose from a strain of V. choleraeO1, serotype Inaba, with identical pattern of resistance to antimicrobians and identical PFGE pattern, suggesting the clonal origin of all isolates (AU)

Salmonella Derby clonal spread from pork.

The genetic diversity of the Derby serotype of Salmonella enterica in Spain was examined by pulsed-field gel electrophoresis (PFGE). Out of 24 identified PFGE profiles, a major clone was detected in 19% of strains from humans, 52% from food, and 62% from swine. This clone (clone 1) was isolated from pork products, suggesting swine as its source.

Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp.

Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens.

Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.