Unmanageable norovirus outbreak in a single resort located in the Dominican Republic.

Noroviruses constitute an important cause of acute gastroenteritis, mainly in semi-closed populations such as hospitals, hotels and cruise ships. This study records the most important norovirus outbreak in the Dominican Republic in a single resort, with more than 800 people being affected in a 15-day period. Analysis of clinical and environmental samples demonstrated that norovirus was the aetiological agent responsible for the outbreak. Although enhanced hygiene and disinfection measures were achieved, the outbreak was only controlled after suspension of entry into the resort.

Gastroenteritis outbreak caused by norovirus associated with the children's club of a hotel located in Majorca, Spain.

A possible gastroenteritis outbreak in a hotel located in northern Majorca was reported on June 2009. The subsequent investigation revealed a total of 14 cases with onset of symptoms from 18 June to 26 June. Symptoms affected mainly the children, their parents and the staff related to the children's club; a vomiting episode was described at the beginning of the outbreak. Genotype 2 norovirus was detected in stool samples, demonstrating its role as the aetiological agent. The special hygienic measures implemented allowed the outbreak to be controlled.

Evaluation of differential gene expression in susceptible and resistant clinical isolates of Klebsiella pneumoniae by DNA microarray analysis.

DNA microarray technology was used to evaluate differential gene expression in a susceptible Klebsiella pneumoniae isolate and a resistant clinical derivative. Nineteen genes were up-regulated in the resistant isolate when compared with the susceptible isolate. An ABC transporter-related gene, ycjV, was strongly over-expressed, suggesting the existence of a novel active efflux mechanism. Approximately half of the up-regulated genes coded for ribosomal proteins, or proteins involved in tRNA metabolism. Among 33 downregulated genes, almost one-third were related to nitrogen metabolism. A possible role of fitness in the development of antimicrobial resistance is suggested.

Identification of two additives, locust bean gum (E-410) and guar gum (E-412), in food products by DNA-based methods.

Locust bean gum (E-410) and guar gum (E-412) are high molecular weight galactomannans used by the food industry as versatile food additives. The compounds, although chemically closely related, do not have the same functional properties when used in foods, and the substitution or unadvertised addition of either could change the desired qualities of the product. Analytical discrimination between E-410 and E-412 is technically difficult since they only differ in their galactose: mannose ratios, being 1 : 4 and 1 : 2 for locust bean gum and guar gum, respectively. A qualitative DNA-based method is reported for the authentication of additives E-410 and E-412 in finished food products (ice cream, dehydrated desserts, milk derivatives, dehydrated soups, salad dressing, marmalade and meat) from small quantities of food. DNA sequences from the nuclear ribosomal spacers of Ceratonia siliqua and Cyamopsis tetragonoloba, the plant sources of E-410 and E-412, respectively, were used to design polymerase chain reaction primers specific for each additive (PA23/PA21 and PG22/PG21). Twenty-two foods were analysed for the presence of E-410 and E-412 additives by this single-step polymerase chain reaction-based method. Positive DNA amplifications with the E-410 and/or E-412 primers were obtained in all 19 samples reported to contain either additive.

Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae.

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three beta-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla(SHV) and bla(TEM) genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, bla(KPC-1), was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing beta-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing beta-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase, KPC-1, although alterations in porin expression may also play a role.

Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18.

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum beta-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single beta-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla(SHV) gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla(SHV)-specific probe indicated that a bla(SHV) gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla(SHV)-related gene revealed that it differs from bla(SHV-1) at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance.

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.

Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniae clinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+ and half O- strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.