scholarly journals A Test For The Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

2021 ◽  
Author(s):  
Sandra Rincon ◽  
Lina P. Carvajal ◽  
Sara I. Gomez-Villegas ◽  
Aura M. Echeverri ◽  
Rafael Rios ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Latin American ◽  
Clinical Laboratory ◽  
Rapid Test ◽  
Reference Value ◽  
Type A ◽  
Reference Standard ◽  
High Inoculum ◽  
Colorimetric Test ◽  
Inoculum Effect

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-β-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin-America and US. Cefazolin MIC determination at high inoculum (107 CFU/mL) was used as reference standard (cut-off of ≥16 μg/mL). All isolates underwent genome sequencing. A total of 257 (37.3%) MSSA exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 μg/mL the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ. The overall negative predictive value ranged from 81.4% to 95.6% in Latin-American countries using published prevalence rates of the CzIE. MSSA from US were genetically diverse, with no distinguishing genomic differences from Latin-American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high-inoculum, the rapid test is inexpensive, feasible and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

scholarly journals Efficacy of Ceftaroline against Methicillin-Susceptible Staphylococcus aureus Exhibiting the Cefazolin High-Inoculum Effect in a Rat Model of Endocarditis

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Kavindra V. Singh ◽  
Truc T. Tran ◽  
Esteban C. Nannini ◽  
Vincent H. Tam ◽  
Cesar A. Arias ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Body Weight ◽  
Infective Endocarditis ◽  
Rat Model ◽  
Type A ◽  
Treated Group ◽  
Content Type ◽  
High Inoculum ◽  
Inoculum Effect

ABSTRACT Certain Staphylococcus aureus strains exhibit an inoculum effect (InE) with cefazolin (CFZ) that has been associated with therapeutic failures in high-inoculum infections. We assessed the in vitro activities of ceftaroline (CPT), CFZ, and nafcillin (NAF) against 17 type A β-lactamase (βla)-producing, methicillin-susceptible S. aureus (MSSA) strains, including the previously reported TX0117, which exhibits the CFZ InE, and its βla-cured derivative, TX0117c. Additionally, we determined the pharmacokinetics of CPT in rats after single intramuscular doses of 20 and 40 mg/kg of body weight and evaluated the activities of CPT (40 mg/kg every 8 h [q8h]), CFZ, and NAF against TX0117 and TX0117c in a rat model of infective endocarditis. No InE was observed for CPT or NAF, whereas a marked InE was detected for CFZ (MIC, 8 to ≥128 μg/ml). CPT and NAF treatment against TX0117 resulted in mean bacterial counts of 2.3 and 2.1 log10 CFU/g in vegetations, respectively, compared to a mean of 5.9 log10 CFU/g in the CFZ-treated group (CPT and NAF versus CFZ, P = 0.001; CPT versus NAF, P = 0.9830). Both CFZ and CPT were efficacious against the βla-cured derivative, TX0117c, compared to time zero (t 0) (P = <0.0001 and 0.0015, respectively). Our data reiterate the in vivo consequences of the CFZ InE and show that CPT is not affected by this phenomenon. CPT might be considered for high-inoculum infections caused by MSSA exhibiting the CFZ InE.

scholarly journals 2176. A New Rapid Test for Detection of The Cefazolin Inoculum Effect (CIE) in Methicillin-Susceptible Staphylococcus aureus (MSSA)

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S738-S739
Author(s):  
Lina P Carvajal ◽  
Karen M Ordonez Diaz ◽  
Edilberto Cristancho Quintero ◽  
Aura M Echeverri ◽  
Sandra Vargas ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Rapid Test ◽  
Type A ◽  
Rapid Identification ◽  
Type B ◽  
High Affinity ◽  
Therapeutic Consequences ◽  
High Inoculum ◽  
Extracellular Form ◽  
Inoculum Effect

Abstract Background Most MSSA harbor one of the four different variants of β-lactamase (BlaZ) (A, B, C and D). The CIE is defined as an MIC >16 μg/mL when a high inoculum (107 CFU/mL) is used and depends on the presence of BlaZ. The presence of the CIE has been associated with therapeutic failure in invasive MSSA infections. In some countries of South America, the prevalence of CIE is high, ranging from 36% to 51% (Colombia and Argentina, respectively). Type A BlaZ is most often associated with the CIE due to its high affinity for cefazolin. Here, we developed a rapid test based on the premise that the extracellular form of BlaZ is responsible for the CIE. We aimed to identify invasive MSSA that exhibit the CIE and validate the test in two cohorts of isolates from patients in Colombia and Argentina Methods 152 MSSA clinical isolates were collected from Colombia (n = 71) and Argentina (n = 81). We determined MIC at standard and high inoculum. We developed a test using induction of BlaZ with ampicillin (150 μg/mL) for 20 minutes and, using the supernatant for incubation with nitrocefin for 30 min. A change in color from yellow to red was considered positive. MSSA TX0117 (BlaZ +, with the CIE), ATCC 29213 (BlaZ-negative) and ATCC 25923 (BlaZ + lacking the CIE) were used as controls. BlaZ typing of all Argentinian isolates was available by sequencing Results A high proportion (43%) of MSSA exhibited the CIE (34% and 52% of Colombian and Argentinian isolates, respectively) by MIC. The rapid test identified 76% of isolates exhibiting the CIE and correctly ruled out all isolates lacking the CIE (sensitivity 80%, specificity 100%). Furthermore, the rapid test detected all isolates with the CIE that harbored Type A BlaZ from Argentina. Conversely, the test failed to identify the CIE in Argentinian isolates that produce type B and C BlaZ. The sensitivity and specificity of the rapid test for the Colombian isolates whose BlaZ type was unknown were 89% and 100%, respectively. Conclusion A rapid test of less than 2 h can readily identify MSSA isolates exhibiting the CIE. For isolates carrying type A BlaZ, which is highly associated with the CIE, the test had a sensitivity and specificity of 100%. Rapid identification of MSSA with the CIE may have important therapeutic consequences in deep-seated infections Disclosures All authors: No reported disclosures.

scholarly journals Inoculum Effect with Cefazolin among Clinical Isolates of Methicillin-Susceptible Staphylococcus aureus: Frequency and Possible Cause of Cefazolin Treatment Failure

2009 ◽  
Vol 53 (8) ◽  
pp. 3437-3441 ◽  
Author(s):  
Esteban C. Nannini ◽  
Martin E. Stryjewski ◽  
Kavindra V. Singh ◽  
Agathe Bourgogne ◽  
Tom H. Rude ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type A ◽  
Hemodialysis Patients ◽  
Clinical Isolates ◽  
Type B ◽  
Exact Test ◽  
Type D ◽  
High Inoculum ◽  
Type C ◽  
Inoculum Effect

ABSTRACT Methicillin (meticillin)-susceptible Staphylococcus aureus (MSSA) strains producing large amounts of type A β-lactamase (Bla) have been associated with cefazolin failures, but the frequency and impact of these strains have not been well studied. Here we examined 98 MSSA clinical isolates and found that 26% produced type A Bla, 15% type B, 46% type C, and none type D and that 13% lacked blaZ. The cefazolin MIC90 was 2 μg/ml for a standard inoculum and 32 μg/ml for a high inoculum, with 19% of isolates displaying a pronounced inoculum effect (MICs of ≥16 μg/ml with 107 CFU/ml) (9 type A and 10 type C Bla producers). At the high inoculum, type A producers displayed higher cefazolin MICs than type B or C producers, while type B and C producers displayed higher cefamandole MICs. Among isolates from hemodialysis patients with MSSA bacteremia, three from the six patients who experienced cefazolin failure showed a cefazolin inoculum effect, while none from the six patients successfully treated with cefazolin showed an inoculum effect, suggesting an association between these strains and cefazolin failure (P = 0.09 by Fisher's exact test). In summary, 19% of MSSA clinical isolates showed a pronounced inoculum effect with cefazolin, a phenomenon that could explain the cases of cefazolin failure previously reported for hemodialysis patients with MSSA bacteremia. These results suggest that for serious MSSA infections, the presence of a significant inoculum effect with cefazolin could be associated with clinical failure in patients treated with this cephalosporin, particularly when it is used at low doses.

scholarly journals Prevalence ofblaZGene Types and the Inoculum Effect with Cefazolin among Bloodstream Isolates of Methicillin-Susceptible Staphylococcus aureus

2012 ◽  
Vol 56 (8) ◽  
pp. 4474-4477 ◽  
Author(s):  
D. J. Livorsi ◽  
E. Crispell ◽  
S. W. Satola ◽  
E. M. Burd ◽  
R. Jerris ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bloodstream Infections ◽  
Fold Increase ◽  
Type A ◽  
Content Type ◽  
High Inoculum ◽  
Inoculum Effect

ABSTRACTWe sought to define the prevalence ofblaZgene types and the inoculum effect to cefazolin among methicillin-susceptibleStaphylococcus aureus(MSSA) bloodstream infections. TheblaZgene was present in 142/185 (77%) isolates. A total of 50 (27%) isolates had a ≥4-fold increase in the cefazolin MIC from a standard to a high inoculum, and 8 (4%) demonstrated a nonsusceptible cefazolin MIC, all type AblaZstrains. The efficacy of cefazolin in the presence of the inoculum effect requires further study.

scholarly journals Prevalence of a Cefazolin Inoculum Effect Associated withblaZGene Types among Methicillin-SusceptibleStaphylococcus aureusIsolates from Four Major Medical Centers in Chicago

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Sheila K. Wang ◽  
Annette Gilchrist ◽  
Anastasia Loukitcheva ◽  
Balbina J. Plotkin ◽  
Ira M. Sigar ◽  
...  
Keyword(s):  
Fold Increase ◽  
Type A ◽  
Content Type ◽  
Microdilution Method ◽  
Medical Centers ◽  
High Inoculum ◽  
Gene Type ◽  
Broth Microdilution Method ◽  
Type C ◽  
Inoculum Effect

ABSTRACTThe efficacy of cefazolin with high-inoculum methicillin-susceptibleStaphylococcus aureus(MSSA) infections remains in question due to therapeutic failure inferred as being due to an inoculum effect (InE). This study investigated the local prevalence of a cefazolin InE (CInE) and its association with staphylococcalblaZgene types among MSSA isolates in the Chicago area. Four medical centers in Chicago, IL, contributed MSSA isolates. Cefazolin MICs (C-MIC) were determined at 24 h by the broth microdilution method using a standard inoculum (SI; 5 × 105CFU/ml) and a high inoculum (HI; 5 × 107CFU/ml). The CInE was defined as (i) a ≥4-fold increase in C-MIC between SI and HI and/or (ii) a pronounced CInE, i.e., a nonsusceptible C-MIC of ≥16 μg/ml at HI. PCR was used to amplify theblaZgene, followed by agarose gel electrophoresis and sequencing to determine the gene type. Approximately 269 MSSA isolates were included. All but one isolate were susceptible to cefazolin at SI, and 97% remained susceptible at HI. A total of 196 isolates (73%) wereblaZpositive, with theblaZtypes led by gene type C (40%). CInE was seen in 45blaZ-positive isolates (23%), with 44 (22%) presenting a ≥4-fold increase in C-MIC (SI to HI) and 5 (3%) a pronounced CInE. Four of the five met both definitions of CInE, two of which expressed the type A gene. The prevalence of a pronounced CInE associated with the type AblaZgene from MSSA isolates in Chicago is low. Our predilection for cefazolin use, even early in the management of hospitalized MSSA infections, is tenable.

scholarly journals 207. High Severity and Mortality Due to Methicillin-Susceptible Staphylococcus aureus Infections in a Colombian Hospital

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S122-S123
Author(s):  
Karen M Ordonez Diaz ◽  
Laura M Baron ◽  
Juan M Garcia ◽  
Edilberto Cristancho ◽  
John Alzate ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Significant Proportion ◽  
Rapid Test ◽  
Source Control ◽  
High Severity ◽  
High Inoculum ◽  
Inappropriate Treatment ◽  
Invasive Infections ◽  
Appropriate Therapy ◽  
The Mean

Abstract Background Methicillin-susceptible Staphylococcus aureus (MSSA) infections are considered less severe than those caused by methicillin-resistant S. aureus. However, we have observed an important increase in severe cases of invasive MSSA infections in a hospital in Pereira, Colombia. Here, we characterize the clinical outcomes and epidemiology of these infections. Methods We included adult and pediatric patients hospitalized between February 2018 and April 2019 presenting with invasive infections caused by MSSA. All isolates were sent to a central laboratory to confirm identification. We determined cefazolin MICs at standard (105 CFU/mL) and high inoculum (107 CFU/mL) by broth microdilution and a rapid test to detect cefazolin inoculum effect (CIE). The CIE was defined as an increase of MIC to ≥16 µg/mL when tested at high inoculum. Clinical data (demographics, intensive care unit (ICU) admission, therapy and mortality) were obtained from medical records. Results A total of 60 patients were included in the study and 41.6% were women. Most (63.3%) infections were hospital-associated. Bacteremia was the most frequent type of infection (71.6%). The mean duration of hospital stay was 24.5 days (IQR, 14–44). 61.6% of patients were admitted to the ICU with a mean length of stay of 14 days (IQR, 7–30). Mortality at 30 days was 28.3% (17 out of 60 patients) and was slightly higher (30.2%) in patients with bacteremia. Early mortality (48 h) was 10% (n = 6). Most patients (75%) received β-lactams (28.8% cefazolin and 84% oxacillin). 18 patients (33%) had isolates that exhibited the CIE but most (n = 11) received oxacillin. Among 17 patients who died, 35% had received antibiotics other than β-lactams (5 vancomycin, 1 ampicillin-sulbactam) and two did not received any therapy due to rapid death. The mean duration of antibiotic therapy was 11 days. Source control was deemed appropriate in 65% of the cases. Conclusion An increase in severe invasive infections caused by MSSA was observed in our hospital with a high proportion of patients requiring ICU care. A significant proportion of patients received inappropriate treatment. Due to the aggressive nature of invasive MSSA infections, efforts to optimize appropriate therapy for these infections are urgently needed in Colombia. Disclosures All authors: No reported disclosures.

scholarly journals Cefazolin high-inoculum effect in methicillin-susceptible Staphylococcus aureus from South American hospitals

2013 ◽  
Vol 68 (12) ◽  
pp. 2773-2778 ◽  
Author(s):  
S. Rincon ◽  
J. Reyes ◽  
L. P. Carvajal ◽  
N. Rojas ◽  
F. Cortes ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
South American ◽  
High Inoculum ◽  
Inoculum Effect

scholarly journals 28. Regulation of the Staphylococcal β-lactamase Plays a Major Role in the cefazolin Inoculum Effect

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S14-S15
Author(s):  
Sara I Gomez-Villegas ◽  
An Q Dinh ◽  
Jinnethe Reyes ◽  
Diana Panesso-Botero ◽  
Maria I Reyes ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Research Study ◽  
Scientific Research ◽  
Regulatory Elements ◽  
Amino Acid Sequences ◽  
Study Investigator ◽  
High Inoculum ◽  
In Trans ◽  
Inoculum Effect ◽  
Unique Amino Acid

Abstract Background Cefazolin (Cz) is commonly used to treat methicillin-sensitive Staphylococcus aureus (MSSA) bacteremia. Yet, some MSSA isolates producing the staphylococcal β-lactamase (BlaZ) exhibit the Cz inoculum effect (CzIE), defined as an increase in the minimum inhibitory concentration (MIC) to ≥ 16 µg/mL at high inoculum (107 CFU/mL, HI-MIC). Retrospective clinical data linked the CzIE to increased 30-day mortality and Cz treatment failure in patients with MSSA bacteremia, yet the mechanistic bases of this phenomenon are unknown. We aimed to explore the contribution of blaZ regulation, via BlaR (antibiotic sensor) and BlaI (transcriptional repressor) (Fig 1) to the CzIE by i) in trans expression assays and ii) analysis of their sequences in a set of isolates Figure 1. Structure of the Staphylococcal bla Operon Methods The blaZ genes (with putative promoters) of strains exhibiting and lacking the CzIE (TX0117 and ATCC29213, respectively) were expressed in trans in RN4220 (blaZ neg) using the promotor-less vector pWM401 (Figure 2). We subsequently cloned the blaR and blaI genes of each TX0117 and ATCC29213 upstream of each blaZ allele (Figure 3). The presence of the CzIE was assessed in transformants using broth microdilution at standard (105 CFU/mL, SI-MIC) and high inoculum. We also performed whole-genome sequencing (WGS) in 104 MSSA isolates exhibiting and lacking the CzIE to compare the sequences of BlaZ, BlaR, and BlaI and classified them by allotypes (unique amino acid sequences) using ATCC29213 as reference. Figure 2. In trans expression of blaZ genes from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Figure 3. In trans expression of the bla Operons from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Results Expression of blaZTX0117 and blaZATCC29213 with their native promoters in RN4220 resulted in the CzIE with Cz HI-MICs ≥ 64 µg/mL regardless of the origin of the allele (Table 1). Inclusion of the regulatory elements blaR and blaI from TX0117 (CzIE+) did not change the phenotype. In contrast, addition of blaR and blaI from ATCC29213 (CzIE-) led to a marked decrease in the Cz HI-MIC (Table 1). Sequence analyses of 104 MSSA isolates revealed 10, 17 and 6 BlaZ, BlaR and BlaI allotypes, respectively (Table 2). BlaZ-2 and BlaR-4 were linked to the CzIE in 90% of isolates. Table 1. MIC values of transformans after In trans expression of blaZ genes and bla Operons from a S. aureus CzIE+ strain(TX0117) and a CzIE- strain (ATCC29213) in RN4220 Table 2. BlaZ allotypes of 104 Staphylococcus aureus isolates and their association with the CzIE Conclusion Our results suggest that overexpression of blaZ can lead to the CzIE in any MSSA strain. Thus, the regulation of blaZ expression via BlaR and BlaI seem to play a major role in the CzIE. Identification of specific BlaR and BlaI allotypes could predict the presence of the CzIE. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

scholarly journals Association between Type AblaZGene Polymorphism and Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

2016 ◽  
Vol 60 (11) ◽  
pp. 6928-6932 ◽  
Author(s):  
Sun Hee Lee ◽  
Wan Beom Park ◽  
Shinwon Lee ◽  
Sohee Park ◽  
Shin Woo Kim ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Single Nucleotide Polymorphisms ◽  
Amino Acid Sequence ◽  
Type A ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Inoculum Concentration ◽  
Content Type ◽  
Inoculum Effect

ABSTRACTSome proportion of type AblaZgene-positive methicillin-susceptibleStaphylococcus aureusstrains exhibit the cefazolin inoculum effect (CIE). The type AblaZgene was divided into two groups by single nucleotide polymorphisms (SNPs) at Ser226Pro and Cys229Tyr. The median cefazolin MICs at a high inoculum concentration were 5.69 μg/ml for the Ser-Cys group and 40.32 μg/ml for the Pro-Tyr group (P= 0.01). The SNPs at codons 226 and 229 in the amino acid sequence encoded by theblaZgene were closely associated with the CIE.

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