Disparate prevalence of toxigenic and nontoxigenic Clostridium difficile among distinct adult patient populations in a single institution.

Clostridium difficile (CD) disease remains a costly and important hospital-associated infection. Although nontoxigenic CD is detected by some CD testing methods, can interfere with some detection algorithms and has been suggested as a treatment for CD disease, little is known about the relative occurrence of toxigenic and nontoxigenic CD in a single institution.We used both chromogenic and selective agar media to recover CD isolates and a molecular method to detect the toxin B gene from over 2400 fresh unformed stool specimens with isolates further tested for the toxin B gene. We recovered 74 nontoxigenic and 306 toxigenic CD isolates for which a collection site could be assigned.The frequency of recovery of toxigenic and nontoxigenic CD for each hospital location and the ratio of toxigenic to nontoxigenic CD were calculated. Although the overall prevalence of toxigenic and nontoxigenic CD was 12.7 % and 3.1 %, respectively, on some wards, 48 % of all CD were nontoxigenic, while on other wards, ≤5 % were nontoxigenic.The disparate ratios of nontoxigenic CD to toxigenic CD presented here for the various 'groups' within the adult veteran population are important to the ongoing discussion and reexamination of other published work on the occurrence of toxigenic and nontoxigenic CD, for evaluating the performance of CD detection tests, for designing infection control strategies and in ultimately understanding both CD carriage and disease.

In Vitro Activity of Retapamulin and Antimicrobial Susceptibility Patterns in a Longitudinal Collection of Methicillin-Resistant Staphylococcus aureus Isolates from a Veterans Affairs Medical Center.

Mupirocin is a topical antimicrobial used to decolonize patients who carry methicillin-resistant Staphylococcus aureus (MRSA), and the topical agent retapamulin may be a potential alternative therapy. The goal of this study was to determine the in vitro activity of retapamulin as well as a panel of 15 antimicrobial agents, including mupirocin, for 403 MRSA isolates collected longitudinally from a naive population at the Veterans Affairs Puget Sound Health Care System. The MICs for retapamulin had a unimodal distribution, ranging from 0.008 to 0.5 µg/ml. One isolate had an MIC of >16 µg/ml, was also resistant to clindamycin and erythromycin, and was recovered from the nares of a patient undergoing hemodialysis. Twenty-four isolates (6%) and 11 isolates (3%) demonstrated low-level resistance (MICs of 8 to 64 µg/ml) and high-level resistance (MICs of ≥ 512 µg/ml), respectively, to mupirocin. Isolates were recovered from 10 patients both before and after mupirocin therapy. Of those, isolates from 2 patients demonstrated MIC changes postmupirocin therapy; in both cases, however, strain typing demonstrated that the pre- and postmupirocin strains were different. A total of 386 isolates (96%) had vancomycin MICs of ≤ 1.0 µg/ml; 340 isolates (84%) were resistant to levofloxacin, 18 isolates (4.5%) were resistant to trimethoprim-sulfamethoxazole, and 135 isolates (33%) had elevated MICs of 4 µg/ml for linezolid. The baseline levels of resistance were low for mupirocin (9%) and even lower for retapamulin (0.25%) Although the use of mupirocin is currently the standard therapy for decolonization practices, the activity of retapamulin warrants its consideration as an alternative therapy in MRSA decolonization regimens.

Clinical significance and characterization of Haemophilus influenzae type b genogroup isolates from urine samples in an adult male population.

The occurrence and significance of Haemophilus spp. isolated from the genitourinary tract are not well known. Herein, we describe the clinical significance and characteristics of Haemophilus influenzae type b genogroup strains isolated from genitourinary tract specimens from an adult male veteran patient population and, in particular, their associations with prostatitis and epididymitis.

Necessity of 16S rRNA gene sequencing for identifying Haemophilus parainfluenzae-like strains associated with opportunistic urinary tract infections.

The identification of Haemophilus spp. from urogenital sites can be challenging due to the lack of appropriate media for culturing the organisms and the poor resolution of biochemical methods. By incorporating chocolate agar and 16S rRNA gene sequence analysis in our protocol to identify Haemophilus spp. from urinary specimens, we isolated and characterized 30 genetically homogeneous strains of a cryptic species that is phylogenetically close to, but distinct from, Haemophilus parainfluenzae. Commercial biochemical kits and VITEK 2 could not distinguish between the two species. Over 90 % of the strains were isolated from urine and the urogenital area, made possible with the inclusion of chocolate agar in our urine culture protocol. In contrast, no Haemophilus strains isolated from respiratory specimens were identified as the cryptic genospecies. The cryptic genospecies was associated with urinary tract infections (UTIs) in certain patient populations. Distinct from Haemophilus quentinii that also causes urogenital infection, the cryptic genospecies required V factor (NAD) but not X factor (haemin) to grow. The data indicated that 16S rRNA gene sequencing may be necessary in identifying Haemophilus species and that inaccurate categorization of Haemophilus strains isolated from urogenital specimens based on phenotypic characteristics may prevent accurate diagnosis of UTIs.

Molecular analysis and clinical significance of Lactobacillus spp. recovered from clinical specimens presumptively associated with disease.

Lactobacillus spp. are part of the normal human flora and are generally assumed to be nonpathogenic. We determined the genotypic identification of >100 Lactobacillus isolates from clinical specimens in the context of presumed pathogenic potential (e.g., recovered as the single/predominant isolate from a sterile site or at ≥10(5) CFU/ml from urine). This study assessed the clinical significance and the frequency of occurrence of each Lactobacillus sp. We identified 16 species of Lactobacillus by 16S rRNA gene sequence analysis, 10 of which could not be associated with disease. While Lactobacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus paracasei were associated with infections, L. gasseri was also a common colonizing/contaminating species. Lactobacillus casei, Lactobacillus johnsonii, and Lactobacillus delbrueckii were associated with at least one infection. Species commonly used in probiotic products (e.g., L. rhamnosus and L. casei) were identical, by 16S rRNA gene sequencing, to our isolates associated with disease. Human isolates of Lactobacillus spp. have differing site associations and levels of clinical significance. Knowing the niche and pathogenic potential of each Lactobacillus sp. can be of importance to both clinical microbiology and the food and probiotic supplement industry.

Identification and clinical significance of Helcococcus species, with description of Helcococcus seattlensis sp. nov. from a patient with urosepsis.

Helcococcus spp. are Gram-positive, catalase-negative, facultatively anaerobic cocci that are associated with wound and prosthetic joint infections as well bacteremia and empyema. Five Helcococcus spp. strains were isolated from our patient population, including 2 strains of Helcococcus kunzii from trauma-associated wounds, 2 Helcococcus sueciensis strains from blood and abscess, and a novel Helcococcus spp. strain from blood associated with urosepsis. Based on the phenotypic and phylogenetic evidence, we propose that the unknown bacterium be classified as Helcococcus seattlensis sp. nov. We found that all 5 tested Helcococcus strains grew as satellite colonies around Staphylococcus aureus and, interestingly, both H. kunzii strains were isolated together with S. aureus. In addition to 16S rRNA gene sequencing, conventional methods for leucine aminopeptidase (LAP) and pyrrolidonyl arylamidase (PYR) testing can be cost-effective and efficient for differentiation of Helcococcus spp. from Abiotrophia and Granulicatella species. Using nonstandard methods, we found that all tested Helcococcus spp. had high MICs of >4/76 µg/ml for trimethoprim-sulfamethoxazole, an antibiotic commonly used to treat urinary tract infections. High MICs for erythromycin, azithromycin, and clindamycin, and intermediate to high MICs for moxifloxacin, levofloxacin, and gentamicin were also observed among the Helcococcus strains.

Impact of identification of Streptococcus dysgalactiae subspecies equisimilis from throat cultures in an adult population.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) are isolated from the throat of patients with pharyngitis, although the clinical significance remains debated. We sought to determine the incidence and association with pharyngitis of SDSE in an adult veteran population. Organisms were phenotypically identified to subspecies and Lancefield group, with selective 16S rRNA gene sequencing. From 833 throat cultures, the overall frequency of SDSE was 3.4% (64% group C and 36% group G) as compared to 8.6% for S. pyogenes (GAS). SDSE was described as a large colony in only 29% of the original culture evaluations by bench technologists, and clinical symptoms were similar for GAS and SDSE. Laboratory algorithms that are limited to identification of only GAS or are based on Lancefield group or visual identification of "large-colony type" ß hemolytic Lancefield group C and G streptococci may be missing or misidentifying SDSE along with Anginosus group streptococci.

Mycotic aneurysm of the abdominal aorta caused by Haemophilus influenzae type f.

Prior to the advent of the Haemophilus influenzae type b vaccine, invasive infections due to H. influenzae type f were rarely described. However, the epidemiology of H. influenzae is changing. While the incidence of invasive infections due to H. influenzae is declining in children, such infections are becoming more common in adults, particularly in the elderly. Here, we report an unusual case of infective aortic aneurysm caused by H. influenzae type f that underlines the emerging clinical relevance and pathogenic capability of this organism.

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates.

The incidence of invasive infections due to Haemophilus influenzae has decreased significantly in developed countries with high rates of vaccination against H. influenzae serotype b (Hib). This vaccine provides no protection against H. influenzae serotype f (Hif), typically associated with invasive infections in adults with chronic disease and/or immunodeficiency, and rarely in otherwise healthy adults and children. The specific properties of Hif associated with virulence remain largely uncharacterized. A panel of 26 Hif strains consisting of both invasive disease-associated and mucosal surface non-invasive disease-associated isolates was surveyed by DNA fingerprinting, biotyping and PCR detection of hmw1, hmw2, hsf, the hif fimbrial locus and the lipo-oligosaccharide (LOS) biosynthetic island, and assessment of ß-lactamase expression and determination of resistance to the bactericidal activity of normal adult human serum. Repetitive sequence-based PCR fingerprinting differentiated the 26 strains into three clusters, with the majority of isolates (22/26, 84.6 %) clustered into a single indistinguishable group. Most isolates (24/26, 92.3 %) were of biotype I and two isolates produced ß-lactamase with detection of a conjugative plasmid, and the isolates displayed a range of resistances to the bactericidal activity of human serum. All 26 isolates carried the adhesin hsf, 21 carried a partial hif fimbrial operon and 4 had the adhesin genes hmw1/2. A LOS biosynthetic island was detected in 20 isolates consisting of the genes lic2BC. It was concluded that Hif has many recognized virulence properties and comprises a relatively homogeneous group independent of the anatomical source from which it was isolated.

Impact of strain typing methods on assessment of relationship between paired nares and wound isolates of methicillin-resistant Staphylococcus aureus.

The anterior nares are the site of choice for the Veterans Administration methicillin-resistant Staphylococcus aureus (MRSA) surveillance program; however, a correlation between nares colonization and concomitant wound infections has not been well established. The purpose of this study was 3-fold: to determine the relatedness of MRSA isolates from 40 paired wound and nares specimens by four different strain typing methods, to determine concordance of typing methods, and to establish a baseline of MRSA types at this medical center. Isolates were typed by repetitive PCR (rep-PCR) (DiversiLab System; DL) and SpectraCell Raman analysis (SCRA) (commercially available methods that can be performed within a clinical lab), pulsed-field gel electrophoresis (PFGE), and an antibiotic susceptibility profile (AB). Whole-genome optical mapping (WGM) (OpGen, Inc.) was performed on selected isolates. All methods agreed that 26 pairs were indistinguishable and four pairs were different. Discrepant results were as follows: 4 where only SCRA was discordant, 3 where only AB was discordant, 2 where both DL and AB were discordant, and 1 where both DL and SCRA were discordant. All WGM agreed with PFGE. After discrepancy resolution, 80% of the pairs were indistinguishable and 20% were different. A total of 56% of nares results were nonpredictive if negative nares and positive wound cultures are included. Methods agreed 85 to 93% of the time; however, congruence of isolates to a clade was lower. Baseline analysis of types showed that 15 pairs were unique to single patients (30 strains, 38%; 47% of the matching pairs). Twenty-five strains (30%) represented a single clade identical by PFGE, SCRA, and DL, decreasing specificity. Typing method and institutional type frequency are important in assessing MRSA strain relatedness.

A lingual abscess caused by Streptococcus intermedius.

Lingual abscesses are rare. We describe a case in a healthy female with no recent history of trauma. The organism recovered by culture of drainage material collected prior to antibiotic treatment was Streptococcus intermedius, an organism recognized as flora of the oropharynx and associated with abscess formation. The isolate was resistant to clindamycin, which was the antibiotic therapy that the patient received.

A longitudinal case series description of meningitis due to Streptococcus gallolyticus subsp. pasteurianus in infants.

Streptococcus gallolyticus subsp. pasteurianus, previously known as Streptococcus bovis biotype II.2, is known to cause multiple infectious complications, including bacterial meningitis, in adults. Only sporadic individual case reports have identified this pathogen as a cause of meningitis in infants. This study is the first to longitudinally document S. gallolyticus subsp. pasteurianus as a cause of meningitis in four epidemiologically unrelated infants less than 2 weeks of age. The 16S rRNA gene sequences of all 4 isolates were identical, and further were identical to 3 central nervous system (CNS) strains (two adults and one child) reported in existing literature. S. gallolyticus subsp. pasteurianus is an increasingly recognized cause of meningitis and bacteremia in the newborn period, and it merits further study with respect to etiology of infection.

Evaluation of a selection strategy before use of 16S rRNA gene sequencing for the identification of clinically significant gram-negative rods and coccobacilli.

Although 16S ribosomal RNA (rRNA) gene sequencing is well established for correctly identifying bacteria, its most efficient use in a routine clinical laboratory is not clear. We devised and evaluated a strategy to select gram-negative rods and coccobacilli (GNRCB) for which sequencing might be necessary before routine identification methods had been exhausted. The prospectively applied selection criteria were primarily based on the isolate's display of unusual or discordant phenotypic results and/or disease correlation. By using this strategy, we selected a total of 120 GNRCB (representing only ∼2% of all identified). The strategy was demonstrated to be efficient because the preliminary phenotypic identification for 79.2% of those isolates needed revision (18.2% were novel and about a third would have required further extensive testing). The knowledge that 1.6% (ie, 79% of 2%) of isolated GNRCB might benefit from sequence identification could provide guidelines for routine clinical laboratories toward efficient use of sequence analysis.

Significantly larger numbers of methicillin-resistant Staphylococcus aureus bacteria are recovered from polymicrobial respiratory and wound sites by use of chromogenic primary media than by use of conventional culture.

Previous studies have validated the properties and documented the utility of chromogenic agar for surveillance of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used one of the chromagars, MRSASelect (Bio-Rad), as one of the primary isolation media for selected wound and respiratory clinical specimens which, in our institution, were typically polymicrobial. We examined a total of 638 specimens; 142 (22%) MRSA isolates were recovered. Twenty-six of these isolates were recovered only on the MRSASelect plate, representing a 28% (15/54) increase for endotracheal aspirates/sputa and a 15% increase for superficial wounds/ulcers (11/73) compared to the results with conventional culture. One isolate (1 CFU) was recovered by conventional medium alone. MRSASelect has generally been used for surveillance cultures; however, we document that an additional 21% of MRSA isolates would have gone unreported in these selected clinical specimens using only standard culture media. For 40% (6/15) of inpatients, MRSA isolated from the MRSASelect plate was the sole indicator of MRSA. Although these isolates can represent either colonization or infection, they are a potential reservoir of infection and nosocomial transmission. Our data support the focused use of chromogenic selective media for the increased detection of MRSA in polymicrobial wound and respiratory specimens, which could have an impact on both clinical treatment and infection control.

Site and clinical significance of Alloscardovia omnicolens and Bifidobacterium species isolated in the clinical laboratory.

Most of the members of the genus Bifidobacterium, including the related organism Alloscardovia omnicolens, are inhabitants of the gastrointestinal tract and oral cavity of humans and animals and have been considered nonpathogenic for humans. However, the actual site of isolation and the clinical significance of A. omnicolens and of Bifidobacterium species are unclear. This may be due in part to the difficulties in distinguishing these organisms from other genera such as Actinomyces. To determine the potential disease-causing role of these organisms, we analyzed the clinical significance of 15 A. omnicolens and Bifidobacterium isolates identified by 16S rRNA gene sequencing from a clinical laboratory. All of the organisms in this study were isolated from sterile sites or in significant numbers by standard clinical microbiological culture methods. Our 15 clinical strains fit into only four species: A. omnicolens (five isolates), Bifidobacterium scardovii (four isolates), B. longum (two isolates), and B. breve (four isolates). All five A. omnicolens isolates, one of the B. breve isolates, and three of the four B. scardovii isolates were cultured from urine at 10(5) CFU/ml. One B. scardovii isolate was from a patient with a genitourinary tract wound infection, two B. longum isolates were from abdominal wounds, and three B. breve isolates were from blood cultures. This study enlarges the spectrum of diseases and clinical sources associated with A. omnicolens and Bifidobacterium species and addresses identification problems.

Thumb infection caused by Streptococcus pseudoporcinus.

Streptococcus pseudoporcinus, a recently described organism found in the genitourinary tract of women, was isolated from a thumb wound in a male patient subsequent to trauma. This case describes a rarely reported non-genitourinary tract clinical isolate of S. pseudoporcinus.

Isolation of Bordetella avium and novel Bordetella strain from patients with respiratory disease.

Bordetella avium is thought to be strictly an avian pathogen. However, 16S rRNA gene sequencing identified 2 isolates from 2 humans with respiratory disease as B. avium and a novel B. avium-like strain. Thus, B. avium and B. avium-like organisms are rare opportunistic human pathogens.

Oral abscess caused by Campylobacter rectus: case report and literature review.

Campylobacter rectus was isolated under routine anaerobic conditions (no additional hydrogen gas in the atmosphere) from an oral, nonperiodontal abscess from a patient with gastroesophageal adenocarcinoma. We report the first case of a palate abscess caused by C. rectus and review the literature and atmospheric requirements of this organism.

Fluoroquinolone use and risk factors for Clostridium difficile-associated disease within a Veterans Administration health care system.

BACKGROUND: Prompted by the changing profile of Clostridium difficile infection and the impact of formulary policies in hospitals, we performed this study when an increase in the incidence of C. difficile-associated disease was noted at our health care center (Veterans Administration Puget Sound Health Care System, Seattle, Washington). METHODS: A retrospective, matched case-control study of patients presenting to the Veterans Administration Puget Sound Health Care System, Seattle, Washington during 2004 was performed. Conditional logistic analysis determined risk factors for case patients, defined as individuals with diarrhea and test results (i.e., culture or toxin assay results) positive for C. difficile, and control subjects, defined as individuals with diarrhea and test results negative for C. difficile. RESULTS: C. difficile-associated disease incidence was 29.2 cases per 10,000 inpatient-days. The increase in the incidence of C. difficile-associated diarrhea that paralleled increased gatifloxacin use was not attributable to use of the antimicrobial but was a reflection of seasonal variation in the rate of C. difficile-associated disease. Multivariate analysis controlling for the time at which the assay was performed, the age of the patient, ward, and source of acquisition (community-acquired vs. nosocomial disease) found 6 significant risk factors for C. difficile-associated diarrhea: receipt of clindamycin (adjusted odds ratio [aOR], 29.9; 95% confidence interval [CI], 3.58-249.4), receipt of penicillin (aOR, 4.1; 95% CI, 1.2-13.9), having a lower intestinal condition (aOR, 2.8; 95% CI, 1.3-6.1), total number of antibiotics received (aOR, 1.4; 95% CI, 1.1-1.7), number of prior hospital admissions (aOR, 1.3; 95% CI, 1.1-1.6), and number of comorbid conditions (aOR, 1.3; 95% CI, 1.1-1.5). CONCLUSIONS: The increase in the number of cases of C. difficile-associated disease was not attributable to a formulary change of fluoroquinolones; instead, the incidence was within expected seasonal variations for C. difficile-associated disease. Recognition of community-acquired cases and the use of culture may help to identify additional cases of C. difficile-associated disease. Early diagnosis and treatment of C. difficile cases may shorten the duration of hospital stays and reduce the number of outbreaks and readmissions, mortality, and other consequences of C. difficile infection.