Continued in vitro cefazolin susceptibility in methicillin-susceptible Staphylococcus aureus.

OBJECTIVES: In vitro trends of cefazolin and ceftriaxone susceptibilities from pediatric clinical isolates of methicillin-susceptible Staphylococcus aureus (MSSA) between 2011 and 2016 were analyzed for surveillance. METHODS: Our laboratory continues to use agar disk diffusion for staphylococcal susceptibilities applying Clinical Laboratory Standard Institute's 2012 breakpoints. RESULTS: A total of 3992 MSSA clinical isolates in the last 6 years were analyzed for their in vitro cefazolin and ceftriaxone susceptibilities. While all MSSA isolates exhibited cefazolin susceptibilities within the "susceptible" zone range, there have been a proportion of isolates with ceftriaxone susceptibilities falling in "intermediate" zones, ranging from 2.6% in 2011 to 8.3% in 2016. CONCLUSIONS: Cefazolin continues to be the recommended agent for MSSA treatment at our institution, reflected by the finding that only 2% (6/321) of patients who received ceftriaxone as definitive therapy for MSSA bacteremia during the study period. We have confirmed the cefoxitin-predicted MSSA susceptibility to cefazolin, but have found concerning drifts in ceftriaxone susceptibilities by continued in vitro monitoring over the last 6 years.

Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples.

Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx1, stx2, and rfbEO157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbEO157, and LD-PCR results prompted successful recovery of E. coli O157 (n = 25) and non-O157 STEC (n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid "mega"-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

Prevalence and mechanisms of broad-spectrum beta-lactam resistance in Enterobacteriaceae: a children's hospital experience.

The objective of this study was to investigate the trends and patterns of resistance in beta-lactamase-producing members of the family Enterobacteriaceae in a children's hospital over a 9-year period (1999 to 2007). Clinically significant isolates of the Enterobacteriaceae were screened for patterns of broad-spectrum resistance to beta-lactams. The strains likely to be resistant were subsequently confirmed by an inhibitor-based disc test. The plasmid-mediated resistance determinants in these isolates were identified by PCR and by in vitro transformation, which successfully reproduced the AmpC phenotype unrestricted by the species of the host organisms. Among 8,048 Enterobacteriaceae isolates belonging to the four chromosomal ampC-negative or -nonfunctional genera, 86 (1.07%) isolates (56 Escherichia coli isolates, 22 Klebsiella species isolates, 1 Proteus mirabilis isolate, and 7 Salmonella species isolates) exhibited broad-spectrum beta-lactam resistance patterns. These organisms collectively produced three classes of beta-lactamases, including class A extended-spectrum beta-lactamases (n = 47), class C or AmpC beta-lactamases (n = 36, including 4 isolates that produced both class A and class C enzymes), and class A or B carbapenem-hydrolyzing beta-lactamases (n = 3). The proportion increased from 0.46% during the first 3 years to 1.84% during the last 3 years (relative risk [RR], 4.04; 95% confidence interval [CI], 2.28 to 7.42; P < 0.001). The increase was mainly due to the emergence of a plasmid-mediated bla(CMY-2) beta-lactamase, the incidence of which increased from 0.11% during the first 3 years to 0.96% during the last 3 years (RR, 9.11; 95% CI, 2.76 to 30.1; P = 0.001). Class A-type resistance increased slightly during the study period, from 0.35% during the first 3 years to 0.85% during the last 3 years (RR, 2.42; 95% CI, 1.15 to 5.07; P = 0.02). A Proteus mirabilis strain was documented to possess a novel bla(DHA) determinant. Of special concern, three carbapenemase-producing isolates were identified between 2003 and 2006. The infections caused by resistant isolates of the Enterobacteriaceae mainly affected hospitalized patients with underlying conditions; however, 19 (22%) episodes were of community onset in otherwise well children. The rate of resistance to broad-spectrum beta-lactams among isolates of the Enterobacteriaceae is increasing in children in both hospital- and community-acquired settings, and the resistance is driven largely by plasmid-mediated AmpC beta-lactamases. These data have important implications for empirical antimicrobial strategies targeting serious pediatric infections. Further study of this problem is warranted.

Characterization of small-colony-variant Stenotrophomonas maltophilia isolated from the sputum specimens of five patients with cystic fibrosis.

Cystic fibrosis (CF) patients are predisposed to chronic respiratory infection by nonfermentative gram-negative bacilli, including Stenotrophomonas maltophilia. S. maltophilia is highly resistant to most antibiotics, with the exception of sulfamethoxazole-trimethoprim (SXT). SXT-resistant S. maltophilia has been reported, but the mechanism of resistance is not well defined. Repeated findings of suspected small-colony-variant (SCV) S. maltophilia isolates from the sputa of five CF patients were confirmed by partial 16S rRNA gene sequencing. The SCV S. maltophilia isolates were the only S. maltophilia isolates in these cultures, and none were clonally related. DNA fingerprint analysis confirmed that once established, the SCV S. maltophilia strains persisted. Nutritional studies of SCV S. maltophilia have suggested auxotrophy in hemin, methionine, and thymidine associated with resistance to multiple antibiotics, including SXT. The phenotypic switch from wild-type to SCV S. maltophilia was reproducible in vitro by exposure to SXT, suggesting that prolonged exposure to antibiotics may select for both the SCV S. maltophilia phenotype and SXT resistance by interference with the dihydrofolate reductase pathway. Recovery of SCV S. maltophilia from the sputum of CF patients has implications for both laboratory testing and patient management.

Ciprofloxacin-resistant gram-negative bacilli in the fecal microflora of children.

The extent to which antibiotic-resistant bacteria are excreted by humans who have not been exposed to antibiotics is not known. Children, who rarely receive fluoroquinolones, provide opportunities to assess the frequency of fecal excretion by fluoroquinolone-naïve hosts of fluoroquinolone-resistant gram-negative bacilli. Fresh nondiarrheal stools from children were processed by screening them on agar containing ciprofloxacin to recover ciprofloxacin-resistant gram-negative bacilli. Resistant isolates were identified, and ciprofloxacin MICs were determined. Resistant Escherichia coli isolates were also analyzed for urovirulence-associated loci. Thirteen (2.9%) of 455 stools yielded ciprofloxacin-resistant E. coli (seven children), Stenotrophomonas maltophilia (four children), and Achromobacter xylosoxidans and Enterobacter aerogenes (one child each). Neither the subjects themselves nor members of their households used fluoroquinolones in the 4 weeks preceding collection. Six of the seven resistant E. coli isolates belonged to phylogenetic groups B2 and D, in which extraintestinal pathogenic E. coli bacteria are frequently found. All resistant E. coli isolates contained at least three putative E. coli virulence loci. Most ciprofloxacin-resistant bacteria were resistant to additional antibiotics. Potentially pathogenic bacteria that are resistant to therapeutically important antimicrobial agents are excreted by some humans, despite these persons' lack of exposure to the particular drugs. The sources of these resistant organisms are unknown. This underrecognized reservoir of drug-resistant potential pathogens poses public health challenges.

Diarrhea etiology in a Children's Hospital Emergency Department: a prospective cohort study.

BACKGROUND: We evaluated the frequency of recovery of pathogens from children with diarrhea who presented to a pediatric emergency department and characterized the associated illnesses, to develop guidelines for performing a bacterial enteric culture. METHODS: We conducted a prospective cohort study of all patients with diarrhea who presented to a large regional pediatric emergency department during the period from November 1998 through October 2001. A thorough microbiologic evaluation was performed on stool specimens, and the findings were correlated with case, physician, and laboratory data. RESULTS: A total of 1626 stool specimens were studied to detect diarrheagenic bacteria and, if there was a sufficient amount of stool, Clostridium difficile toxin (688 specimens), parasites (656 specimens), and viruses (417 specimens). One hundred seventy-six (47%) of 372 specimens that underwent complete testing yielded a bacterial pathogen (Shiga toxin-producing Escherichia coli, 39 specimens [of which 28 were serotype O157:H7]; Salmonella species, 39; Campylobacter species, 25; Shigella species, 14; and Yersinia enterocolitica, 2), a viral pathogen (rotavirus, 85 specimens; astrovirus, 27; adenovirus, 18; or rotavirus and astrovirus, 8), a diarrheagenic parasite (5 specimens); or C. difficile toxin (46 specimens). Samples from 2 patients yielded both bacterial and viral pathogens. A model to identify predictors of bacterial infection found that international travel, fever, and the passing of >10 stools in the prior 24 h were associated with the presence of a bacterial pathogen. Physician judgment regarding the need to perform a stool culture was almost as accurate as the model in predicting bacterial pathogens. CONCLUSIONS: Nearly one-half of the patients who presented to the emergency department with diarrhea had a definite or plausible pathogen in their stool specimens. We were unable to develop a model that was substantially better than physician judgment in identifying patients for whom bacterial culture would yield positive results. The unexpectedly high rate of C. difficile toxin warrants further examination.

Etiology of diarrhea in pediatric outpatient settings.

BACKGROUND: The frequency with which bacteria cause diarrhea evaluated in ambulatory settings is often unknown. We attempted to determine the microbiologic etiology of diarrhea in a private pediatric practice (site A) and a clinic serving largely immigrant children (site B) and to establish guidelines for bacterial culture. METHODS: Children with diarrhea were prospectively enrolled, and their stools were examined for diarrheagenic bacteria, viruses and parasites. RESULTS: A total of 123 and 103 children were enrolled at sites A and B, respectively. Stools from all (100%), 126 (55.8%), 104 (46.0%) and 75 (33.2%) were tested for bacterial enteric pathogens, parasites, Clostridium difficile toxin and viruses, respectively. Of the 75 patients whose stool underwent complete testing, 36 (48%) contained at least 1 definitive or plausible pathogen. Twelve stools (5.3%) tested positive for bacteria [Campylobacter jejuni (n = 7), Yersinia enterocolitica, Shigella flexneri, Shigella sonnei, Salmonella serogroup D and Salmonella Braenderup (n = 1 each)]. One contained Blastocystis hominis, 8 contained C. difficile toxin and 16 contained viruses (9 rotavirus, 5 adenovirus and 2 astrovirus). Visible fecal blood (P = 0.029), increased stool frequency (P = 0.035), abdominal tenderness (P = 0.011) and fecal white (P < 0.001) or red blood cells (P = 0.002) were associated with bacterial infection. All children with stool yielding diarrheagenic bacteria or C. difficile toxin had at least 1 of these factors, but so did 75% of children without these agents (positive predictive value, 11%; negative predictive value, 100%; sensitivity, 100%; specificity, 25%). CONCLUSIONS: The bacterial diarrhea prevalence is similar to that in other ambulatory studies, although the spectrum differs. Exclusion criteria for stool testing in diarrhea remain elusive. Studies to determine the etiology of unexplained diarrhea and cost-effective algorithms for diarrhea diagnosis, are needed.

Shiga toxin-producing Escherichia coli in children with diarrhea: a prospective point-of-care study.

OBJECTIVE: To conduct a prospective cohort study to determine the frequency and characteristics of Shiga toxin (Stx)-producing Escherichia coli (STEC) infections in children with diarrhea attending an emergency department and a private clinic in Seattle, Washington. METHODS: Between November 1998 and October 2001, 1851 stools were processed for STEC by sorbitol-MacConkey (SMAC) agar screening and a commercial Stx enzyme immunoassay (EIA). RESULTS: STEC belonging to serotypes O157:H7 (n = 28), O103:H2 (n = 4), O118:H16 (n = 2), O26:H11, O111:nonmotile, O111:H8, O121:H19, and O rough:H11 (n = 1 each) were recovered from 39 (2.1%) stools. EIA and SMAC agar detected 89% and 100% of the patients with E coli O157:H7, respectively. E coli O157:H7-infected patients had significantly higher frequencies of bloody stools, fecal leukocytes, and abdominal tenderness and shorter symptom duration. Hemolytic uremic syndrome developed in 5 (18%) and none of the children infected with E coli O157:H7 and non-O157:H7 STEC, respectively (P =.30). CONCLUSIONS: E coli O157:H7 is the predominant STEC in this population. Children infected with E coli O157:H7 have clinical presentations different from those whose stools contain non-O157:H7 STEC. Culture and Stx detection are needed to optimally detect STEC of all serotypes in stools. SMAC agar screening should not be replaced by EIA.