Microbial recovery from clot-activating Vacutainers®.

Biological specimens for microbiological analysis are often collected in BD Vacutainers®, which are not specifically designed for microbial recovery. Bacterial and fungal recovery was analyzed for glass and plastic tubes with or without clot-activating silica. No significant impact was found for the recovery of most bacteria and yeasts tested, however, Haemophilus influenzae recovery from cerebrospinal fluid was significantly reduced in both glass and plastic clot activator tubes.

In Vitro Antimicrobial Susceptibility of Staphylococcus pseudintermedius Isolates of Human and Animal Origin.

MIC results for 115 Staphylococcus intermedius group isolates are presented. Of these, 33% were methicillin resistant, among which 51.4% were susceptible to doxycycline, 29.7% to clindamycin, and 21.6% to trimethoprim-sulfamethoxazole. All of the isolates were susceptible to ceftaroline, daptomycin, linezolid, nitrofurantoin, quinupristin-dalfopristin, rifampin, tigecycline, and vancomycin. Of all the isolates, 82.6%, 67.8%, and 23.5% were susceptible to ciprofloxacin, erythromycin, and penicillin, respectively. No isolates harbored mupA or qacA/B genes, which suggested a lack of resistance to mupirocin or chlorhexidine.

Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory.

This study compared the diagnostic performance of Bruker's Microflex LT and bioMérieux's Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. A total of 477 isolates were tested on both instruments. Discrepant results were resolved by sequencing. Overall, there was no statistically significant difference between the proportion of isolates correctly identified, miscalled or not called by each instrument. Although both systems were good at identifying yeast (66/69 to species level), the confidence level was high only to genus level for 30% of the isolates on the Bruker. Both systems performed with high accuracy when evaluated solely on Food and Drug Administration-approved organisms for each database. A user-based assessment of the 2 instruments revealed an overall preference for the Vitek MS instrument.

Surveillance for enteric pathogens in a case-control study of acute diarrhea in Western Kenya.

BACKGROUND: Acute diarrhea remains a major public health problem in East African nations such as Kenya. Surveillance for a broad range of enteric pathogens is necessary to accurately predict the frequency of pathogens and potential changes in antibiotic resistance patterns. METHOD: Stool samples were collected from September 2009 to September 2011; 193 and 239 samples, from age-matched cases and asymptomatic controls, were collected, respectively, from Kericho and Kisumu District Hospitals in western Kenya. Bacterial pathogens were identified by conventional microbiological methods; antibiotic susceptibility of bacterial isolates was ascertained using the MicroScan WalkAway 40 Plus. An enzyme immunoassay kit was used to detect rotavirus, and ova and parasite examination was conducted by microscopy and an enzyme immunoassay. RESULTS: Rotavirus (10.2% and 10.5%) and Shigella (11% and 8%) were isolated significantly more often in the cases than the controls from Kericho and Kisumu District Hospitals respectively. The diarrheagenic Escherichia coli, Campylobacter jejuni and Salmonella were found most often in the cases while Giardia lamblia and Entamoeba histolytica/E. dispar were found more often in the controls. Most pathogens were isolated from children under 5 years old. More than 50% of the Shigella, Salmonella and diarrheagenic E. coli isolates were multidrug resistant to ampicillin, tetracycline and trimethoprim/sulfamethoxazole with several enteroaggregative and enterotoxigenic E. coli isolates producing extended-spectrum beta-lactamases. CONCLUSION: Accurate epidemiologic information on acute diarrheal illness in Kenya will be critical for augmenting existing diarrhea management policies in terms of treatment and to strengthen future community awareness and health promotion programs.

Exposure to rumen protozoa leads to enhancement of pathogenicity of and invasion by multiple-antibiotic-resistant Salmonella enterica bearing SGI1.

Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that has been purported to be more virulent than antibiotic-sensitive counterparts. The paradigm for this multiresistant/hyperpathogenic phenotype is Salmonella enterica serotype Typhimurium phage type DT104 (DT104). The basis for the multiresistance in DT104 is related to an integron structure designated SGI1, but factors underlying hyperpathogenicity have not been completely identified. Since protozoa have been implicated in the alteration of virulence in Legionella and Mycobacterium spp., we attempted to assess the possibility that protozoa may contribute to the putative hypervirulence of DT104. Our study reveals that DT104 can be more invasive, as determined by a tissue culture invasion assay, after surviving within protozoa originating from the bovine rumen. The enhancement of invasion was correlated with hypervirulence in a bovine infection model in which we observed a more rapid progression of disease and a greater recovery rate for the pathogen. Fewer DT104 cells were recovered from tissues of infected animals when protozoa were lysed by preinfection chemical defaunation of the bovine or ovine rumen. The protozoan-mediated hypervirulence phenotype was observed only in DT104 and other Salmonella strains, including serovars Agona and Infantis, possessing SGI1.

SlyA regulates the collagenase-mediated cytopathic phenotype in multiresistant Salmonella.

Salmonella enterica serotype Typhimurium phagetype DT104 (DT104) is a foodborne pathogen with a multiresistant phenotype conferred by a genomic-based integron structure designated as SGI1. Recently, a novel cytopathic phenotype was ascribed to several isolates of DT104 recovered from veal calves. This phenotype is dependent upon clg, a gene encoding a collagenase in Salmonella. Using a novel transposon system and an RT-PCR assay for detection of clg expression, we identified SlyA as a regulator of the collagenase-mediated phenotype. The function of SlyA, in regards to clg expression, is to repress the synthesis of Clg. Derepression ensued in the absence of SlyA or in the presence of a truncated version of SlyA with the latter being relevant for maintenance of another virulence aspect mediated by SlyA, i.e. survival within macrophages. The SlyA-mediated effect on clg expression was restricted to DT104 and other Salmonella phagetypes and serotypes possessing SGI1 thus suggesting co-regulation by an SGI1-specific component.

Avoidance of false PCR results with the integron-retron junction in multiple antibiotic resistant Salmonella enterica serotype Typhimurium.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Another concern with this pathogen is the ability to acquire integrons that confer resistance to multiple antibiotics. For multiresistant Salmonella enterica serotype Typhimurium, the most common multiresistant Salmonella serotype, an integron structure can be found between thdF and a retron. Our objective was to investigate the utility of a 450 bp thdF-retron amplicon as an indicator of an insertless thdF-retron junction thus indicating an integron-free strain. Surprisingly, we found that the 450 bp thdF-retron amplicon was present, and thus incorrectly suggesting an integron-free status, in some multiresistant S. enterica serotype Typhimurium isolates. However, this phenomenon was not observed if the isolate was enriched in the presence of two antibiotics. This demonstrates that, within some individual clinical isolates of multiresistant S. enterica serotype Typhimurium, there exists a small subpopulation of integron-free bacteria. Consequently, it appears that the thdF-retron amplicon is an inaccurate predictor of integron status in S. enterica serotype Typhimurium unless multiresistance is used as a selection tool during enrichment.

Cytopathic effects observed upon expression of a repressed collagenase gene present in Salmonella and related pathogens: mimicry of a cytotoxin from multiple antibiotic-resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Recently, we reported that certain strains of Salmonella enterica serovar Typhimurium phagetype DT104 (DT104) secrete a putative cytotoxin. While searching for the gene that encodes this toxin, we noted a previously reported but uncharacterized DNA fragment (clg) in Salmonella that could be potentially relevant to cytotoxin-like activity. Therefore, we cloned and expressed clg in cytotoxin-negative Escherichia coli and Salmonella and subsequently assessed the bioactivity of Clg in vitro and in vivo. Lysates containing Clg from both expression hosts exerted cytopathic effects on murine enterocytes while semi-purified Clg was determined to be cytopathic to HEp-2 cells. Sequence and RT-PCR analyses of the clg gene indicated that a homologue of clg exists in different Gram-negative bacteria although the gene is not expressed in vitro. Although Clg-mediated lesions are similar to those mediated by the DT104 cytotoxin, further investigations are necessary to examine the relationship between these two proteins in DT104. Nonetheless, we report here a defined Salmonella protein that is capable of inflicting damage on tissue culture cells and murine enterocytes.

A high-throughput genetic system for assessing the inhibition of proteins: identification of antibiotic resistance and virulence targets and their cognate inhibitors in Salmonella.

This study describes the development of a high-throughput genetic system for producing oligopeptides that can be used to identify molecular interactions leading to inhibition of specific proteins. Using a pathogenic bacteria model, we screened a library of clones expressing intracellular oligopeptides in order to identify inhibitors of proteins involved in antibiotic resistance and virulence. This method involved transforming the pathogen with an oligopeptide-encoding plasmid library, constructed using polymerase chain reaction and an oligonucleotide template designed to produce random oligopeptides composed of 2-16 amino acids, and high-throughput screening for phenotype alterations in the pathogen. A subsequent complementation phase enabled the identification of the full-length bacterial protein inhibited by the oligopeptide. Using this method we were able to identify oligopeptides that inhibit virulence and/or drug resistance in Salmonella, Shigella, and Escherichia coli; specific virulence and/or drug resistance proteins of Salmonella, Shigella, and E. coli that are sensitive to inhibition; and putative oligopeptide-binding sites on the inhibited proteins. This system is versatile and can be extended to other pathogens for analogous studies and it can be modified for used in eukaryotic models for identifying protein interactions that can be targeted for inhibition. Additionally, this system can be used for identifying protein domains involved in any biomolecular interaction.