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The aim of this research was to investigate the potential relationship, if any, between the acquisition/possession of antibiotic resistance genes in strains of Salmonella and its resistance to heat stress. Chicken pieces were inoculated with antibiotic sensitive (AS) strains of S. Enteritidis and S. Typhimurium, its laboratory-acquired antibiotic-resistant (AR) mutant strains (nalidixic acid and streptomycin), or a multiresistant strain of S. Typhimurium DT104. Half of these samples were heat-shocked (48C for 30 min) and all were heat-challenged at 55C for up to 30 min. Samples were then plated on xylose lysine desoxycholate (XLD) and tryptone soya agar (TSA) overpoured with XLD. Heat-shocked cultures of S. Typhimurium DT104 had significantly higher D-values (the time required for a 1 log reduction in the number of bacteria) than their non-heat-shocked counterparts (P [1] 0.05). No significant differences were observed between AR and their AS. However, the D-values for S. Typhimurium DT104 were significantly higher than the D-values for S. Typhimurium (AS) and S. Enteritidis (AS) (P [1] 0.05). This study concluded that laboratory-acquired antibiotic-resistant mutation did not affect heat resis-tance of the Salmonella strains studied and suggested a potential link between multiantibiotic resistance and heat resistance in S. Typhimurium DT104.

Rotavirus is the primary etiological agent of gastroenteritis in infants and young children worldwide. In developing countries, it is estimated that rotavirus is responsible for one-third of all diarrhea-associated hospitalizations and 873,000 deaths annually. In industrialized countries, where mortality from rotavirus is low, infection is widespread, and nearly all children experience an episode of rotavirus diarrhea in the first 3-5 yr of life. Rotaviruses have important antigenic specificities including serogroup and serotype, and all viruses are classified accordingly. They are divided into seven morphologically indistinguishable but antigenically defined serogroups, delineated A through G. The human infecting rotaviruses include groups A, B, and C, and it is well documented that group A rotaviruses are the major causative agents of diarrheal diseases in children. They are responsible for 125 million cases of diarrhea annually. Within each serogroup, distinct serotypes exist. In group A rotavirus, serotype is specified by two viral proteins, VP4 and VP7. The neutralizing antibody response that is evoked by the antigenic determinants on VP4 and VP7 play an important role in protective immunity. The rotavirus genome consists of 11 double-stranded (ds) RNA segments, and each genomic segment encodes a different protein. A dual system of reporting rotavirus serotype exists because the VP4 and VP7 proteins are encoded by different genes and thus can segregate independently. The serotypes derived from VP7 are defined as G-serotypes. Currently 14 G-serotypes have been identified, and only 10 of these have been recovered from humans. The predominating G-types worldwide are G1, G2, G3, and G4, with G1 being the most prevalent type. Serotypes G5, G6, G8-G10, and G12 are rarely identified in humans and are usually recovered from animals. However, some of these unconventional types are now being frequently reported in humans, including G5, G8, and G9.

Antimicrobial resistance determinants may be transferred among bacteria via mobile genetic elements including plasmids, transposons, and the more recently explored integrons. Integrons are naturally occurring genetic elements found as part of the Tn21 transposon family or located on various broad host-range plasmids. The fundamental integron structure consists of a 5'-conserved segment (5'-CS) of 1.4-kbp and a 2-kbp 3'-CS. Between these conserved regions are DNA sequences of variable length and molecular complexity. These intervening sequences are known as gene cassettes, and several have now been characterized. Acquisition and dissemination of these genes located within the integron structure, results in an increase in antimicrobial resistance. Three classes of integron structure have been described. Class 1 integrons are of principal importance in clinical isolates. The 5'-CS of class 1 integrons includes an intI 1 gene of 1358 bp, which encodes a specific recombinase, a member of the DNA integrase family. This gene contains the att1 recombination site, required for specifically integrating gene cassettes. Classes 2 and 3 also contain integrase genes (intI 2 and intI 3), with the former showing 40% sequence identity to those of class 1, and the latter showing 61%. All three classes of integrons contain similar gene cassettes from the same families, which suggests the existence of a common pool of gene cassette with cross-specificity between the classes. When the 3'-CS region is examined in detail, it contains several open reading frames (ORFs). These include qacEDelta1, which confers resistance to quaternary ammonium compounds, often associated with antiseptics, along with a sul1 gene expressing resistance to sulphonamide antimicrobial agents. The sul1 gene encodes the enzyme hydopteroate synthase. Transcription of the sul1 gene begins at a promoter located in the 5'-CS. The latter is also responsible for the transcription of the inserted gene cassette(s). Two additional ORFs, ORF-5 and -6, are located toward the distal end of the 3'-CS. The gene product of ORF 5 appears to share some sequence similarity with puromycin acetyltransferase, and this feature suggests a possible role in antimicrobial resistance. A biological function has yet to be ascribed to ORF-6.

Thermophilic Campylobacter spp., mainly Campylobacter jejuni and to a lesser extent C. coli are recognized as the most common bacteriological causes of gastroenteritis in humans. As enteric infection with Campylobacter organisms cannot be distinguished from that caused by other enteric pathogens, a definitive diagnosis can only be made by isolating or detecting the organism from the feces. The epidemiology of Campylobacter enteritis has been complicated by the ubiquitous nature of the organism (commonly found as a commensal in the intestines of domestic animals, in milk, and in water). Furthermore, identification is carried out only to genus level by most clinical laboratories. Because of the biochemical similarity known to exist between C. jejuni and C. coli, the hippurate hydrolysis test is often used as the only phenotypic test capable of differentiating the two species. This test, however, has some acknowledged technical limitations and is dependent on inoculum size; results can be difficult to interpret accurately. Furthermore, almost all C. jejuni isolates possess the hippuricase gene, fewer C. jejuni isolates express the hippuricase gene. For this reason, certain polymerase chain reaction (PCR)-based species identification methods, for both C. jejuni and C. coli, and for the other thermophilic species, provide more reliable identification; they also help to highlight mixed species cultures, should they occur. However, even with these methods, false negatives or nonspecifically amplified product(s) can occur in a minority of isolates tested owing to genomic anomalies. Thus a second molecular identification method may be required in these circumstances. Gonzalez et al. developed a species-specific PCR assay for the identification of C. jejuni and C. coli based on the ceuE gene, which is involved in siderophore transport. Using this method two primer sets are employed in separate PCR amplification reactions. Another method, developed by Eyers et al., performs PCR amplification of 23S rRNA gene fragments, based on regions specific for C. jejuni, C. coli, C. lari, and C. upsaliensis. In addition, Hani and Chan developed a PCR assay that detected and amplified the hippuricase gene. This molecular approach may offer a more reliable means of identifying C. jejuni strains compared with the phenotypic hippurate hydrolysis test alone.

Rotavirus is the primary etiological agent of gastroenteritis in infants and young children worldwide. In developing countries, it is estimated that rotavirus is responsible for one-third of all diarrhea-associated hospitalizations and 873,000 deaths annually. In industrialized countries, where mortality from rotavirus is low, infection is widespread, and nearly all children experience an episode of rotavirus diarrhea in the first 3-5 yr of life. Rotaviruses have important antigenic specificities including serogroup and serotype, and all viruses are classified accordingly. They are divided into seven morphologically indistinguishable but antigenically defined serogroups, delineated A through G. The human infecting rotaviruses include groups A, B, and C, and it is well documented that group A rotaviruses are the major causative agents of diarrheal diseases in children. They are responsible for 125 million cases of diarrhea annually. Within each serogroup, distinct serotypes exist. In group A rotavirus, serotype is specified by two viral proteins, VP4 and VP7. The neutralizing antibody response that is evoked by the antigenic determinants on VP4 and VP7 play an important role in protective immunity. The rotavirus genome consists of 11 double-stranded (ds) RNA segments, and each genomic segment encodes a different protein. A dual system of reporting rotavirus serotype exists because the VP4 and VP7 proteins are encoded by different genes and thus can segregate independently. The serotypes derived from VP7 are defined as G-serotypes. Currently 14 G-serotypes have been identified, and only 10 of these have been recovered from humans. The predominating G-types worldwide are G1, G2, G3, and G4, with G1 being the most prevalent type. Serotypes G5, G6, G8-G10, and G12 are rarely identified in humans and are usually recovered from animals. However, some of these unconventional types are now being frequently reported in humans, including G5, G8, and G9.

Thermophilic Campylobacter spp., mainly Campylobacter jejuni and to a lesser extent C. coli are recognized as the most common bacteriological causes of gastroenteritis in humans. As enteric infection with Campylobacter organisms cannot be distinguished from that caused by other enteric pathogens, a definitive diagnosis can only be made by isolating or detecting the organism from the feces. The epidemiology of Campylobacter enteritis has been complicated by the ubiquitous nature of the organism (commonly found as a commensal in the intestines of domestic animals, in milk, and in water). Furthermore, identification is carried out only to genus level by most clinical laboratories. Because of the biochemical similarity known to exist between C. jejuni and C. coli, the hippurate hydrolysis test is often used as the only phenotypic test capable of differentiating the two species. This test, however, has some acknowledged technical limitations and is dependent on inoculum size; results can be difficult to interpret accurately. Furthermore, almost all C. jejuni isolates possess the hippuricase gene, fewer C. jejuni isolates express the hippuricase gene. For this reason, certain polymerase chain reaction (PCR)-based species identification methods, for both C. jejuni and C. coli, and for the other thermophilic species, provide more reliable identification; they also help to highlight mixed species cultures, should they occur. However, even with these methods, false negatives or nonspecifically amplified product(s) can occur in a minority of isolates tested owing to genomic anomalies. Thus a second molecular identification method may be required in these circumstances. Gonzalez et al. developed a species-specific PCR assay for the identification of C. jejuni and C. coli based on the ceuE gene, which is involved in siderophore transport. Using this method two primer sets are employed in separate PCR amplification reactions. Another method, developed by Eyers et al., performs PCR amplification of 23S rRNA gene fragments, based on regions specific for C. jejuni, C. coli, C. lari, and C. upsaliensis. In addition, Hani and Chan developed a PCR assay that detected and amplified the hippuricase gene. This molecular approach may offer a more reliable means of identifying C. jejuni strains compared with the phenotypic hippurate hydrolysis test alone.

Objectives . To employ a combination of phenotypic and genotypic subspecies typing methods to aid in an epidemiological investigation of an outbreak of Salmonella bredeney involving ten persons. Methods . Isolates were characterised by employing antibiogram typing, in addition to two genotyping techniques, including pulsed field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD) with two oligonucleotide primers. Results . An outbreak of gastroenteritis associated with S. bredeney (serovar O:4 H:Lv 1,7) occurred in Belfast, Northern Ireland in November 1997. In total, ten cases were confirmed, of which eight had consumed chicken cooked at local butchers and retailed through one of two local bakeries. One of the remaining cases was secondarily infected within her home and the final case had eaten a product other than cooked chicken from one of the bakeries. Food preparation practices were inadequate in one of the bakeries in question and record keeping and possibly cooking procedures were inadequate in the butchers. S. bredeney was isolated from an uncooked chicken supplied to the butchers confirming that improperly cooked chicken was most likely the source of the outbreak. All outbreak clinical isolates were indistinguishable from each other and were similar to the isolate obtained from the uncooked poultry demonstrating that these DNA-based methods were valuable in the molecular characterization of S. bredeney . Conclusions . This report emphasises the importance and maintenance of an effective hazard analysis critical control point (HACCP) approach to the processing and retailing of foodstuffs containing chicken in order to help eliminate hazards to public health.

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Definition of the diversity and antimicrobial susceptibility of S. sonnei isolates may be helpful in the management of individual cases and outbreaks. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of S. sonnei predominantly ( n = 59) from three counties in the west of Ireland. Phage typing ( n = 17), plasmid profiling ( n = 28), and integron analysis ( n = 24) were performed with subsets of strains. PFGE typing permitted recognition of two major clusters: PFGE type A ( n = 53) and PFGE type B ( n = 14). PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed ( n = 6) were phage type 3. PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates). Fifteen different plasmid profiles were identified among the 28 isolates analyzed. A class 2 integron was present in all 14 PFGE type B isolates. One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern. Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin. Our data demonstrate two predominant PFGE types among S. sonnei isolates circulating in this region. The limited diversity of the S. sonnei isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak. Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.

Measurements were made of the susceptibility to six commonly prescribed antibiotics, including erythromycin, tetracycline and ciprofloxacin, of 130 isolates of Campylobacter jejuni and 15 isolates of Campylobacter coli cultured from human and poultry sources during 2000. The results were compared with the results from a collection of strains isolated between 1996 and 1998. The levels of resistance to erythromycin remained low, 2 per cent and 4.4 per cent for the human and poultry isolates, respectively. Resistance to tetracycline had increased to 31 per cent and 24.4 per cent from 13.9 per cent and 18.8 per cent for the human and poultry isolates, respectively. However, the resistance to ciprofloxacin of the strains isolated during 2000 had increased to 30 per cent, whereas between 1996 and 1998 there had been no resistance to this agent among human isolates, and only 3-1 per cent resistance among poultry isolates. The molecular basis for this resistance has been shown to be the result of a single amino acid substitution, Thr-86-lle, in the gyrA subunit of DNA gyrase in C jejuni . A subset of 59 isolates was tested by molecular methods and all of the 25 phenotypically resistant isolates possessed this substitution. None of the human isolates had been treated with ciprofloxacin before their laboratory isolation.

A restriction fragment length polymorphism (RFLP) detection assay was developed to examine the genetic relationship(s) among VP7-encoding genes from 100 Irish rotavirus isolates and 30 randomly selected global rotavirus isolates (from the current databases). RFLP analysis of the VP7 gene segments was performed independently with three enzymes ( Rsa I, Alu I, and Eco RV) in separate reactions by direct digestion of the DNA product amplified by reverse transcriptase (RT)-mediated PCR (RT-PCR) or by using computational methods. Thirty-six RFLP patterns were identified for all 130 strains, and of these, only nine patterns were associated with the Irish isolates. A correlation between the G type of the Irish isolates and certain single or combined enzyme profiles was apparent. These data suggested that the Irish wild-type rotavirus population was homogeneous and could be distinguished by RFLP analysis from global isolates of the same serotype(s). The deduced amino acid sequences of the VP7 RT-PCR products from six Irish isolates known to be of the G serotype revealed significant amino acid substitutions within major antigenic regions. In addition, these data identified the existence of at least two genetic lineages within serotype G1 strains which were distinguishable by RFLP analysis.

A recombinant Rhizobium meliloti β-galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p -nitrophenyl β-D-galactopyranoside (PNPG) and o -nitrophenyl β-D-galactopyranoside (ONPG) with K m PNPG and K m ONPG of 1 mM at 25°C. The k cat / K m ratios for both substrates were approximately 70 mM −1 sec −1 , indicating no clear preference for either PNPG or ONPG, unlike E. coli β-galactosidase. After non-denaturing electrophoresis, active β-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl β-D-galactopyranoside (BNG) and diazo blue B.

Two hundred and twenty-six Salmonella enterica serotype Typhimurium isolates were examined for the presence of integron-associated gene cassettes. All but two of the non-DT104 isolates, together with DT104 isolates, contained gene cassettes. Amplicons of 1.5 kbp each were found in two non-DT104 isolates, encoding a dhfrI gene (trimethoprim resistance) linked to an aadA gene (streptomycin and spectinomycin resistance), by site-specific recombination. DT104 isolates of resistance (R) type ACSSuT possessed the recently described 1.0- and 1.2-kbp gene cassettes. Macrorestriction analysis with Xba I and DNA probing mapped ant ( 3" )- 1a , bla PSE-1 , and dhfrI genes to large multiresistant gene clusters in a DT170a isolate and a DT193 isolate. In contrast, all DT104 isolates (R-type ACSSuT) possessed a conserved 10-kbp Xba1 DNA fragment. Our study highlights the occurrence of integrons (and antimicrobial resistance determinants) among serotype Typhimurium isolates other than DT104. Larger and previously unrecognized multiresistance gene clusters were identified in these isolates by DNA probing.

A collection of three hundred thirty rotavirus-positive stool samples from children with diarrhea in the southern and eastern regions of Ireland between 1997 and 1999 were submitted to the Molecular Diagnostics Unit of the Cork Institute of Technology, Cork, Ireland, for investigation. These strains were characterized by several methods, including polyacrylamide gel electropherotyping and G and P genotyping. A subset of the G types was confirmed by nucleic acid sequencing. The most prevalent types found in this collection included G1P[8] (n = 106; 32.1%), G2P[4] (n = 94; 28.5%), and G4P[8] (n = 37; 11.2%). Novel strains were also detected, including G1P[4] (n = 19; 5.8%), and G4P[4] (n = 2; 0.6%). Interestingly, mixed infections accounted for 18.8% (n = 62) of the total collection, with only 3% (n = 10) which were not G and/or P typeable. Significantly, six G8 and five G9 strains were identified as part of mixed infections. These strains have not previously been identified in Irish children, suggesting a greater diversity in rotavirus strains currently circulating in Ireland.

Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica . In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated ( n = 48). Phage types DT104b ( n = 3), -193 ( n = 9), -195 ( n = 6), -208 ( n = 3), -204a ( n = 2), PT U302 ( n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.

SummarySimultaneous fluorescent [F] detection of the factor V Leiden (G1691A) and the prothrombin 3’-untranslated region (G20210A) mutations were performed in a single tube polymerase chain reaction (PCR). Amplification refractory mutation detection system (ARMS) formed the basis of this assay design. Fluorescent-labelled primers incorporated into amplicons during the reaction facilitated detection directly by GeneScan analysis without further manipulation. To test the efficacy of this double [F]-ARMS (dF-ARMS) method, 48 patients with unexplained thrombotic tendencies were investigated for their factor V Leiden and prothrombin genotypes. These results corresponded exactly with data achieved using the more conventional methods of restriction fragment length polymorphism (RFLP)-PCR and direct DNA sequencing. Three out of the 48 patients in this group were found to be compound heterozygotes.

To the Editor: Clostridium difficile is the most common cause of bacterial diarrhea in hospitalized patients (1). Antimicrobial drug therapy is the most important risk factor associated with the acquistion of C. difficile, and several antimicrobial agents including clindamycin, amoxicillin, and cephalosporins have been particularly associated with C. difficile infection (2). Acquisition of resistance to clindamycin is considered 1 mechanism whereby clonal strains emerge and predominate in healthcare environments (3). Historically, fluoroquinolone antimicrobial agents were considered low risk for C. difficile–associated-disease; however, recent studies indicate a shift in the risk associated with their use (4). Furthermore, recent outbreaks in Canada and the United States have been associated with fluoroquinolone exposure (4). Recently, several C. difficile outbreaks due to PCR ribotype 027 (PCR-027) and associated with increased disease severity and death have been reported worldwide (4). This strain type contains the genes for binary toxin and has an 18-bp deletion and a frameshift mutation in tcdC hypothesized to result in deregulated expression of toxins A and B. These strains produce 16× more toxin A and 23× more toxin B in vitro than toxinotype 0 strains (5). These isolates demonstrate universal high-level resistance to fluoroquinolones in contrast to that of PCR 027 isolates collected before 2001 (4). We report the mechanism of fluoroquinolone resistance in a cluster (n = 5) of Irish PCR-027 C. difficile isolates that were characterized by using toxinotyping and 16–23S ribotyping. Amplification with PCR and sequencing was used to identify the binary toxin gene (cdtB) and an 18-bp deletion and a frameshift mutation at position 117 in the tcdC gene. Antimicrobial susceptibility to 5 fluoroquinolone antimicrobial drugs was determined with E-tests (AB-Biodisk, Solna, Sweden). The quinolone-resistance–determining region (QRDR) of gyrA and gyrB was amplified by PCR and characterized. The nucleotide sequence data for partial sequences of the gyrA gene were submitted to GenBank and assigned accession nos. {"type":"entrez-nucleotide","attrs":{"text":"DQ821481","term_id":"118638607","term_text":"DQ821481"}}DQ821481, {"type":"entrez-nucleotide","attrs":{"text":"DQ821482","term_id":"118638609","term_text":"DQ821482"}}DQ821482, {"type":"entrez-nucleotide","attrs":{"text":"DQ821483","term_id":"118638611","term_text":"DQ821483"}}DQ821483, and {"type":"entrez-nucleotide","attrs":{"text":"DQ821484","term_id":"124365596","term_text":"DQ821484"}}DQ821484. PCR ribotyping profiles identified 1 cluster of C. difficile PCR-027 with clinical isolates that showed indistinguishable profiles to the control 027 strain. PCR identified the cdtB, an 18-bp deletion, and a frameshift mutation at position 117 in the tcdC gene in all 5 isolates. These strains were universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin, respectively, MIC >32 μg/mL [Table]). Control isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3, 0.2 μg/mL, respectively); however, these strains had reduced susceptibility to levofloxacin (MIC 3 μg/mL) and were resistant to ciprofloxacin and ofloxacin (Table). Sequence analysis determined that all 5 PCR-027 isolates had a single transition mutation (C to T), resulting in the amino acid substitution Thr-82-Ile in gyrA (Table). No amino acid substitutions were found in the QRDR of gyrB (data not shown). Table Characterization of representative isolates, Ireland, 2006 Mutations in the active site or the QRDR of DNA gyrase and topoisomerase IV have been associated with increased resistance to fluoroquinolones in several bacteria (6). This report identifies for the first time a mutation in gyrA that is associated with high-level resistance to fluoroquinolones in C. difficile PCR-027. In Escherichia coli, amino acid substitutions that occur at Ser-83 in gyrA have been associated with fluoroquinolone resistance (6). Thr-82 in C. difficile corresponds to Ser-83 in E. coli. Thr-to-Ile amino acid substitutions corresponding to Ser-83 have been associated with fluoroquinolone resistance in several bacteria, including Pseudomonas aeruginosa, Enterobacter aerogenes, Campylobacter jejuni, and C. difficile (6). Ackermann et al. described 2 mutations in gyrA that resulted in an amino acid substitution corresponding to codon 83 in E. coli. Thirteen of the 18 C. difficile isolates had the Thr-82-Ile substitution, and 1 strain had a Thr-82-Val substitution (7). Dridi et al. described this Thr-82-Ile GyrA substitution in 6 resistant C. difficile strains corresponding to 3 serogroups, H1, A9, and 1C (8). Early studies investigating fluoroquinolone antimicrobial agents suggested that most C. difficile isolates were susceptible to these drugs. Antimicrobial drug resistance to this class has increased with fluoroquinolone use, and currently these drugs remain the most frequently prescribed antimicrobial agents in the United States and Europe. Acquired resistance to the newer fluoroquinolone antimicrobial agents is not restricted to ribotype PCR-027, although different amino acid substitutions in the QRDR of gyrA and gyrB have been described (7–9). Wilcox et al. have described high-level fluoroquinolone resistance in PCR ribotype-001, an endemic strain type found in several healthcare settings in the United Kingdom (10). We have previously described the emergence of a fluoroquinolone-resistant toxin A–, toxin B–positive strain in Dublin (9). We report a mutation in gyrA associated with fluoroquinolone resistance in C. difficile PCR-027. Antimicrobial drug resistance in C. difficile isolates must be monitored because the emergence of universal fluoroquinolone resistance in different C. difficile strain types may be a factor promoting outbreaks in hospitals. As exposure to several different fluoroquinolone antimicrobial drugs have been independently associated with C. difficile–associated-disease, restricted use of all fluoroquinolones, rather than changing from 1 quinolone to another, may be a necessary step toward preventing and controlling C. difficile outbreaks.

1. This study investigated the potential of a commercially available acidified water treatment (PWT) for reducing the number of Campylobacter in vitro and other bacteria in the gut of live broilers. 2. In vitro tests indicated that PWT was highly effective for reducing Campylobacter jejuni and Campylobacter coli at the recommended concentration in water, reducing populations by greater than 7 log10 CFU/ml after 24 h exposure. The decrease in the number of Salmonella serovar Enteritidis and Escherichia coli was not significant. 3. Addition of PWT to the broiler drinking water for the first 7 d, 2 d before and 2 d after each feed change and at feed withdrawal prior to slaughter or only after feed withdrawal had no effect on the number of Campylobacter in caecal samples on farm before thinning and depopulation compared to untreated controls. 4. Although PWT was effective for reducing Campylobacter in water, the results suggest that it does not reduce the number of Campylobacter in the caeca of broilers prior to slaughter under the conditions used in the study.

Guidelines on prudent antimicrobial use in veterinary medicine have been developed to reduce inappropriate prescribing of antimicrobials. Such guidelines focus mainly on the clinical and pharmacological indications for prescribing. A questionnaire study of veterinary surgeons engaged in cattle practice was completed to determine if non-clinical issues influence the decision to prescribe antimicrobials, and to assess if pharmacological and non-pharmacological issues influence the choice of antimicrobial prescribed. Non-clinical issues, including issues related to professional stress, influenced the prescribing decision of the majority of respondents. However, the nature of the veterinarian-client relationship did not influence the prescribing behaviour of the majority of respondents. Pharmacological and non-pharmacological issues influenced the choice of antimicrobial prescribed. The veterinary surgeon's prior experience of a drug was considered 'often' or 'always' by 95.7 per cent of respondents when making this decision. The findings of this study have implications for the recognition and management of stress within the profession, and for the development of intervention strategies to reduce inappropriate antimicrobial prescribing.

Screening biological samples using the polymerase chain reaction (PCR) has obvious advantages compared with current molecular analytical methods based on gel electrophoresis and/or hybridisation, both of which are expensive and time-consuming, therefore the development of a PCR assay format that is applicable to large sample numbers and that can readily use equipment commonly found in diagnostic laboratories would be advantageous. This report describes the development of a colour amplified PCR detection system which is simple in design and could be universally applied to the detection of any DNA template. As an example, the system has been applied in the detection of the heat-stable toxin coding gene (ST-gene) from enterotoxigenic Escherichia coli (ETEC). The assay is sensitive, detecting 10 fg of a purified DNA template and 270 cfu of an ST-gene-positive ETEC strain.

This paper reports on three applications of the polymerase chain reaction (PCR) in the pathology laboratory, cystic fibrosis carrier status investigation, Staphylococcus aureus identification and HLA-DQ alpha tissue typing. Allele specific PCR was used to detect the common cystic fibrosis mutation (delta F508) in an Irish family. The genomic status of each member was shown to be either heterozygous (carrier) or homozygous (affected) for the mutation, based on the resolution of allelic amplifications in an agarose gel. Five staphylococci were subjected to PCR designed to amplify a 137 bp DNA fragment from the S. aureus protein A gene (spa). Four isolates gave a coloured "dot-blot" positive signal, the fifth was negative. In the final application the HLA-DQ alpha region was amplified using DNA obtained from four unrelated individuals. Amplified DNA was tissue typed in a reverse "dot-blot" format. All individuals had unique HLA-DQ alpha types. These examples have been chosen to demonstrate the versatility of this technique and to illustrate some of its potential applications.

We read with interest the letter of Armstrong et al. (D. Armstrong, S. Bell, M. Robinson, P. Bye, B. Rose, C. Harbour, C. Lee, H. Service, M. Nissen, M. Syrmis, and C. Wainwright, Letter, J. Clin. Microbiol. 41:2266-2267, 2003) describing the occurrence of a clonal type (pulsotype I) of Pseudomonas aeruginosa at five cystic fibrosis (CF) centers along the east coast of Australia. This letter strongly asserts that this clonal type has spread to CF patients, not from either the hospital or external environments, but by person-to-person transmission. This letter also describes some of the recent events that have led to advances in molecular typing techniques and thus to the identification of a dominant and hypertransmissible clonal strain of P. aeruginosa at several CF centers. What this letter also demonstrates to the CF microbiology community is that there is an urgent requirement for the standardization and harmonization of genotyping techniques, both nationally and internationally, where several methods presently exist for strain characterization (for a detailed review of P. aeruginosa typing, see reference 15). Armstrong et al. (letter) cite several previous studies employing pulsed-field gel electrophoresis (PFGE) typing of P. aeruginosa. A methodological comparison highlighting technical variations in previous PFGE typing studies of P. aeruginosa is detailed in Table ​Table1.1. Thus, Armstrong et al. were correct and diligent in comparing interlaboratory variability in deciding the relatedness of isolates from five CF centers, when more than one microbiology laboratory was involved in strain analysis. TABLE 1. Comparison of PFGE typing methodologies Recently Jones et al. (9) have stressed the importance of microbiological surveillance through molecular fingerprinting, namely, PFGE typing, in order to ascertain the extent of cross-infection of P. aeruginosa among CF patients. PFGE is no longer a method solely restricted to epidemiological typing research studies, but is one which is now used in many clinical diagnostic laboratories to aid with routine epidemiological analysis. Laboratories which are introducing such techniques for the analysis of their P. aeruginosa subtypes should adopt protocols that will yield data for their isolates which can be compared with data from other CF centers. This strategy has been successfully applied for typing enteric bacterial pathogens through the PulseNet system (www.cdc.gov/pulsenet). The exchange of typing data obtained by employing standardized and harmonized methods, via an electronic medium, as has been developed recently by the ESF Network for Exchange of Microbial Typing Information (http://lists.nottingham.ac.uk/mailman/listinfo/enemti), would facilitate our understanding of the molecular epidemiology of P. aeruginosa in CF patient populations. Consensus is thus needed in order to ascertain if such differences, for example, different suppliers of restriction enzymes, internal standards, pulse field parameters, etc., are significant or not. If the effects of such differences are not known, then a feasibility study involving several centers needs to be undertaken to resolve these technical anomalies, so that a robust method may be adopted by the CF microbiology community. If it felt that such a trial is unnecessary, then it will be a relatively easy task to define a consensus PFGE protocol that will gain widespread acceptance. The real treasure to the CF microbiology community of adopting standardized and harmonized genotyping protocols such as PFGE is that it will allow us to identify transmissible types locally as well as globally and provide a basis for the examination of virulence in common genotypes. If we fail to attempt to learn a form of “molecular Esperanto” in terms of speaking a common language of harmonized and standardized methodology, we will potentially suffer, as befell the Israelites in Old Testament times with the Tower of Babel (Genesis 11), from misunderstanding. Indeed, if we cannot communicate with each other, we can hardly cooperate with each other.

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin Bpositive (A – B + ) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A – B + strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified daily by review of ELISA results and were cultured on selective media. Following culture, toxin-specific immunoassays, IMR-90 cytotoxicity assays and PCR were used to analyse consecutive C. difficile isolates from 93 patients. Using a toxin A-specific ELISA, 52 samples produced detectable toxin. All isolates were positive using a toxin A ⁄ B ELISA. Similarly, all isolates were positive with the cytoxicity assay, although variant cytopathic effects were observed in 41 cases. PCR amplification of the toxin A and toxin B genes revealed that 41 of the previous A – B + strains had a c. 1.7-kb deletion in the 3¢-end of the tcdA gene. Restriction enzyme analysis of these amplicons revealed the loss of polymorphic restriction sites. These 41 A – B + isolates were designated toxinotype VIII by comparison with C. difficile strain 1470. PCR ribotyping revealed that all A – B + isolates belonged to PCR-ribotype 017. A – B + C. difficile isolates accounted for 44% of the isolates examined in this study, and appeared to be isolated more frequently in Dublin, Ireland, than reported rates for other countries.

The Rhizobium meliloti (Rm) lacZ gene provides a convenient model to investigate patterns of gene regulation in these agronomically important bacteria. A gene encoding beta-galactosidase (beta Gal) activity was cloned from R. meliloti by complementing a lactose-negative Escherichia coli mutant. A series of Sau3A subclones was generated in pBR322, and the coding region for the beta Gal-coding gene was localized to a 2.4-kb core fragment. In E. coli 'maxicells', these lacZ subclones produced a 79-kDa polypeptide, irrespective of the fragment size demonstrating that the translation initiation signal(s) are located on the 2.4-kb fragment. Transposon Tn5 mutagenesis and BAL 31 deletion analysis showed that the expression of the Rm lacZ gene in E. coli was dependent on the tetracycline-resistance promoter of pBR322. The cloned sequence was required for beta Gal synthesis in Rhizobium since mutants generated by reverse genetics lack this enzyme and were specifically defective in lactose catabolism.

Whereas exposure of combinations of a phenothiazine and bacterium to incoherent UV increases the activity of the phenothiazine, exposure of the phenothiazine alone does not yield an increase of its activity. Because the laser beam energy is greater than that produced by the incoherent UV sources, exposure of phenothiazines to specific lasers may yield molecules with altered activities over that of the unexposed parent. Chlorpromazine, thioridazine and promethazine active against bacteria were exposed to two distinct lasers for varying periods of time. Absorption and fluorescence spectra were conducted prior to and post-exposure and the products of laser exposure evaluated for activity against a Staphylococcus aureus ATCC strain via a disk susceptibility assay. Exposure to lasers alters the absorption/fluorescence spectra of the phenothiazines; reduces the activity of thioridazine against the test bacterium; produces a highly active chlorpromazine compound against the test organism. Exposure of phenothiazines to lasers alters their structure that results in altered activity against a bacterium. This is the first report that lasers can alter the physico-chemico characteristics to the extent that altered bioactivity results. Exposure to lasers is expected to yield compounds that are difficult to make via chemical manipulation methods. A survey of selected patents of interest, even co-lateral for the subject of this article is shortly made.

Enterococcus faecalis is recognized as a multidrug-resistant nosocomial pathogen. The phenotypic basis for this is largely uncharacterized. The intrinsic efflux system of the antibiotic-susceptible E. faecalis ATCC29212 strain was studied using a semi-automated method that assesses accumulation and efflux of the universal efflux pump substrate ethidium bromide (EB). The results show that the intrinsic efflux system of this Enterococcus strain is controlled by energy derived from the catabolism of glucose and the proton concentration of the medium. At pH 5, agents that inhibit efflux pumps in Gram-positive organisms and the proton gradient un-coupler CCCP do not increase accumulation nor inhibit efflux of EB. In contrast, at pH 8, where the proton concentration is 1,000-fold lower, these agents increase accumulation and efflux of EB. These results are relevant to infections produced by E. faecalis and subsequent antibiotic therapy with antibiotics to which the organism is known to be intrinsically resistant.

Salmonella Typhimurium is the predominant serotype isolated from humans in Europe. Pork and pork products are recognized vehicles of Salmonella and are responsible for outbreaks of human salmonellosis. Pigs can become infected with Salmonella on the breeding or fattening farm and during transport, lairage, and slaughter. The aim of this study was to investigate selected points of Salmonella contamination from the time pigs entered the lairage to the time the carcass was processed in the boning hall and to determine the importance of different sources of Salmonella along the Irish pork production chain. A second objective was to evaluate whether the serological status or category of a herd influenced the levels of bacteriological contamination detected on individual carcasses and pork cuts during slaughter and dressing operations. All samples were tested for the presence and numbers of Salmonella. Enterobacteriaceae numbers were also determined. Serotype, phage type, and pulsed-field gel electrophoresis were utilized to determine similarity among Salmonella isolates. Lairage was a major source of cross-contamination with Salmonella as were the hands of evisceration operatives, conveyor belts, and equipment in the boning hall. Cross-contamination within the slaughter plant environment accounted for up to 69 % of Salmonella carcass contamination. In general, herd category reflected the bacteriological status of carcasses and pork cuts. Major findings were a strong association (P < 0.01) between Enterobacteriaceae counts and Salmonella occurrence on prechill carcasses and a significant association (P < 0.05) between Enterobacteriaceae counts and Salmonella occurrence on pork cut samples.

Phenothiazines have their primary effects on the plasma membrane of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources, energy providing enzymes such as ATPases, and genes that regulate and code for permeability aspects of the bacterium. The responses of multi-drug (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis to the neuroleptic phenothiazine thioridazine are reviewed. The information collated suggests that this phenothiazine has the potential to cure XDR and MDR tuberculosis infections, a potential that has been recently demonstrated by its ability to cure 10 patients who presented with XDR TB infections. The mechanism by which this phenothiazine produces the desired effects within the infected macrophage is also discussed.

In the developing world, enterotoxigenic Escherichia coli (ETEC) strains which produce enterotoxins are a significant cause of morbidity and mortality. Heat-labile (LT) toxin PCR detection methods have been described, but they have limited applications in a routine laboratory setting. A colorimetric DNA method for the rapid amplification and detection of the LT toxin gene in ETEC strains is described. Target amplification together with colorimetric detection would overcome many of the limitations of conventional PCR. This paper describes a colorimetric PCR detection method specific for LT-gene-encoding ETEC strains. DNA was extracted from two representative colonies from each bacterial isolate and amplified by PCR. Digoxigenin was incorporated into the amplification product, permitting a one-step direct detection using anti-digoxigenin alkaline phosphatase-conjugated antibody. This technique was applied to the investigation of 70 E. coli isolates derived from clinical fecal samples obtained from an Irish population. Eleven percent of the samples were LT positive, confirming the applicability of this method. All LT-positive ETEC strains (controls and clinical isolates) were detected, and no false-positive results occurred.

We report the first documentation of C. difficile 027 in Ireland from a patient with diarrhoea transferred from a hospital in the United Kingdom (UK). In addition, we report two clusters of C. difficile ribotype 027 in two hospitals in Ireland.

The aim of the present study was to evaluate a new one-step enrichment protocol, consisting of a combined preenrichment and enrichment broth (Cronobacter Enrichment Broth [CEB]) used in conjunction with selective-differential agar ChromID Sakazakii, to facilitate a shortened 2-day cultural method for detection of Cronobacter spp. (Enterobacter sakazakii) in powdered infant formula (PIF). The CEB was evaluated using samples artificially inoculated with low concentrations of 10 lyophilized strains, representative of the genus Cronobacter. The detection of strains was compared in parallel with the enrichment medium from ISO/TS 22964 and a recently proposed differential screening broth for the detection of Cronobacter. All of the Cronobacter strains were recovered using the CEB, and a significantly higher final bacterial concentration was obtained with the CEB than with the other enrichment broths (P < 0.01). There was no significant difference between the cell concentrations for cultures grown in CEB at 37 degrees C and those grown at 41.5 degrees C. Cronobacter was recovered from both 1/10 (50 g:450 ml) and 1/5.5 (100 g:450 ml) sample-to-broth ratios, with no significant difference observed between the final bacterial concentrations obtained from the two ratios. Further studies on a wider range of PIFs, including naturally contaminated samples, are warranted to determine if the use of this protocol may facilitate the rapid release (within 40 to 48 h) of PIF.

http://aem.asm.org/ Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.

Bacterial multidrug resistance may be mediated by the overexpression of efflux pumps. Conventional evaluation of efflux activity using efflux pump substrates, such as ethidium bromide, requires specialised instrumentation. The agar-based method, previously reported, has been modified to evaluate as many as twelve bacterial strains and has been termed the ethidium bromide-agar cartwheel method.Agar plates containing different concentrations of ethidium bromide were swabbed with bacterial cultures. The cell efflux capacity increased with increasing ethidium bromide concentration, which produced fluorescence of the bacterial mass.The method was shown to be useful for the detection of efflux activity among multidrug-resistant Gram-negative and Gram-positive clinical isolates, as confirmed by the determination of minimum inhibitory concentration for several antibiotics in the presence of known efflux pump inhibitors.This method may be adapted to the clinical laboratory for the presumptive identification of multidrug-resistant isolates that overexpress efflux pump systems.

The genetic similarity of Campylobacter jejuni isolates from pets, compared to human clinical cases and retail food isolates collected in Ireland over 2001-2006 was investigated by cluster analysis of pulsed-field gel electrophoresis (PFGE) fingerprinting profiles. Comparison of the PFGE profiles of 60 pet isolates and 109 human isolates revealed that seven (4.1%) profiles were grouped in clusters including at least one human and one pet C. jejuni isolate. In total six (1.6%) of 60 pet and 310 food profiles were in clusters with at least one food and one pet C. jejuni isolate. The detection of only a small number of genetically indistinguishable isolates by PFGE profile cluster analysis from pets and from humans with enteritis in this study suggests that pets are unlikely to be an important reservoir for human campylobacteriosis in Ireland. However, genetically indistinguishable isolates were detected and C. jejuni from pets may circulate and may contribute to clinical infections in humans. In addition, contaminated food fed to pets may be a potential source of Campylobacter infection in pets, which may subsequently pose a risk to humans.

Background A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction. Results The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method. Conclusion Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.

Cryptosporidiosis, a protozoal disease which causes significant morbidity in humans, is one of the chief causes of diarrhoea in neonatal ruminants. although the parasite poses a significant threat to public health and animal health in Ireland, its epidemiology on the island is only poorly understood. Environmental studies have shown the waterborne parasite to be widespread in some untreated waterbodies around Ireland. The island's hydrogeological situation, combined with high stocking rates of livestock and the absence of filtration from regular water treatment, render it vulnerable to large-scale outbreaks. This review discusses the parasite in the Irish context and underlines the need for a reference facility to provide active surveillance on the island.