Prevalence of the cefazolin inoculum effect (CzIE) in nasal colonizing methicillin-susceptible Staphylococcus aureus in patients from intensive care units in Colombia and use of a modified rapid nitrocefin test for detection.

The cefazolin inoculum effect (CzIE) has been associated with poor clinical outcomes in patients with methicillin-susceptible Staphylococcus aureus (MSSA) infections. We aimed to investigate the point prevalence of the CzIE among nasal colonizing MSSA isolates from ICU patients in a multicenter study in Colombia (2019-2023). Patients underwent nasal swabs to assess for S. aureus colonization on admission to the ICU, and some individuals had follow-up swabs. We performed cefazolin MIC by broth microdilution using standard and high inoculum and developed a modified nitrocefin-based rapid test to detect the CzIE. Whole-genome sequencing was carried out to characterize BlaZ types and allotypes, phylogenomics, and Agr-typing. A total of 352 patients were included; 46/352 (13%) patients were colonized with S. aureus and 22% (10/46) and 78% (36/46) with MRSA and MSSA, respectively. Among 36 patients who contributed with 43 MSSA colonizing isolates, 21/36 (58%) had MSSA exhibiting the CzIE. BlaZ type A and BlaZ-2 were the predominant type and allotype in 56% and 52%, respectively. MSSA belonging to CC30 were highly associated with the CzIE, and single-nucleotide polymorphism (SNP) analyses supported possible transmission of MSSA exhibiting the CzIE among some patients of the same unit. The modified nitrocefin rapid test had 100%, 94.4%, and 97.7% sensitivity, specificity, and accuracy, respectively. We found a high point prevalence of the CzIE in MSSA colonizing the nares of critically ill patients in Colombia. A modified rapid test was highly accurate in detecting the CzIE in this patient population.

Inoculum effect of CTX-M-15, OXA-48, and KPC-2 producing Klebsiella pneumoniae on meropenem and ceftazidime-avibactam efficacy.

The inoculum effect, characterized by diminished antibacterial activity at high bacterial inocula, is studied in the context of beta-lactam and beta-lactamase inhibitor combinations against beta-lactamase-producing Enterobacterales. The inhibition of ESBL + OXA-48 and KPC enzymes, in combination with ceftazidime, demonstrates encouraging results. In this study, 20 Klebsiella pneumoniae isolates were tested with different inocula (1-5 × 105 and 1-5 × 107 cfu/ml) using broth microdilution methods. The inoculum effect was observed in meropenem against OXA-48 + CTX-M-15- and KPC-2-producing isolates but not with ceftazidime/avibactam. Notably, meropenem exhibited inoculum effect against carbapenemase-producing strains, whereas ceftazidime-avibactam remained effective. We conclude that ceftazidime-avibactam is recommended for high-inoculum infections.

Inoculum effect of antimicrobial peptides.

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 105 cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.

The Influence of Heteroresistance on Minimum Inhibitory Concentration, Investigated Using Weak-Acid Stress in Food Spoilage Yeasts.

Populations of microbial cells may resist environmental stress by maintaining a high population-median resistance (IC50) or, potentially, a high variability in resistance between individual cells (heteroresistance); where heteroresistance would allow certain cells to resist high stress, provided the population was sufficiently large to include resistant cells. This study sets out to test the hypothesis that both IC50 and heteroresistance may contribute to conventional minimal inhibitory concentration (MIC) determinations, using the example of spoilage-yeast resistance to the preservative sorbic acid. Across a panel of 26 diverse yeast species, both heteroresistance and particularly IC50 were positively correlated with predicted MIC. A focused panel of 29 different isolates of a particular spoilage yeast was also examined (isolates previously recorded as Zygosaccharomyces bailii, but genome resequencing revealing that several were in fact hybrid species, Z. parabailii and Z. pseudobailii). Applying a novel high-throughput assay for heteroresistance, it was found that IC50 but not heteroresistance was positively correlated with predicted MIC when considered across all isolates of this panel, but the heteroresistance-MIC interaction differed for the individual Zygosaccharomyces subspecies. Z. pseudobailii exhibited higher heteroresistance than Z. parabailii whereas the reverse was true for IC50, suggesting possible alternative strategies for achieving high MIC between subspecies. This work highlights the limitations of conventional MIC measurements due to the effect of heteroresistance in certain organisms, as the measured resistance can vary markedly with population (inoculum) size. IMPORTANCE Food spoilage by fungi is a leading cause of food waste, with specialized food spoilage yeasts capable of growth at preservative concentrations above the legal limit, in part due to heteroresistance allowing small subpopulations of cells to exhibit extreme preservative resistance. Whereas heteroresistance has been characterized in numerous ecological contexts, measuring this phenotype systematically and assessing its importance are not encompassed by conventional assay methods. The development here of a high-throughput method for measuring heteroresistance, amenable to automation, addresses this issue and has enabled characterization of the contribution that heteroresistance may make to conventional MIC measurements. We used the example of sorbic acid heteroresistance in spoilage yeasts like Zygosaccharomyces spp., but the approach is relevant to other fungi and other inhibitors, including antifungals. The work shows how median resistance, heteroresistance, and inoculum size should all be considered when selecting appropriate inhibitor doses in real-world antimicrobial applications such as food preservation.

Comparison of the inoculum effect of in vitro antibacterial activity of Imipenem/relebactam and Ceftazidime/avibactam against ESBL-, KPC- and AmpC-producing Escherichia coli and Klebsiella pneumoniae.

OBJECTIVE: To evaluate effect of inoculum size of extended-spectrum ß-Lactamase (ESBL)-producing-, AmpC-producing-, and KPC-producing Escherichia coli and Klebsiella pneumoniae on the in vitro antibacterial effects of imipenem/relebactam (IMR) and ceftazidime/avibactam (CZA). METHODS: We compared the impact of inoculum size on IMR and CZA of sixteen clinical isolates and three standard isolates through antimicrobial susceptibility tests, time-kill assays and in vitro PK/PD studies. RESULTS: When inoculum size increased from 105 to 107 CFU/mL, an inoculum effect was observed for 26.3% (5/19) and 52.6% (10/19) of IMR and CZA, respectively; time-kill assays revealed that the concentration of CZA increased from ≥ 4 × MIC to 16 × MIC to reach 99.9% killing rate against K. pneumoniae ATCC-BAA 1705 (KPC-2-, OXA-9- and SHV-182-producing) and 60,700 (SHV-27- and DHA-1-producing). While for IMR, a concentration from 1 × MIC to 4 × MIC killed 99.9% of the four strains. When the inoculum size increased to 109 CFU/mL, neither IMR nor CZA showed a detectable antibacterial effect, even at a high concentration. An in vitro PK/PD study revealed a clear bactericidal effect when IMR administered as 1.25 g q6h when inoculum size increased. CONCLUSION: An inoculum effect on CZA was observed more frequent than that on IMR. Among the ß-lactamase-producing strains, the inoculum effect was most common for SHV-producing and KPC-producing strains.

Stochastic bacterial population dynamics restrict the establishment of antibiotic resistance from single cells.

A better understanding of how antibiotic exposure impacts the evolution of resistance in bacterial populations is crucial for designing more sustainable treatment strategies. The conventional approach to this question is to measure the range of concentrations over which resistant strain(s) are selectively favored over a sensitive strain. Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying resistance plasmids, we show that single resistant cells have <5% probability of detectable outgrowth at antibiotic concentrations as low as one-eighth of the resistant strain's minimum inhibitory concentration (MIC). This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, well below the MIC of resistant strains, may effectively restrict de novo emergence of resistance even though they cannot clear already-large resistant populations.

A New Pharmacokinetic-Pharmacodynamic Model To Characterize the Inoculum Effect of Acinetobacter baumannii on Polymyxin B In Vitro.

The inoculum effect (i.e., reduction in antimicrobial activity at large starting inoculum) is a phenomenon described for various pathogens. Given that limited data exist regarding inoculum effect of Acinetobacter baumannii, we evaluated killing of A. baumannii by polymyxin B, a last-resort antibiotic, at several starting inocula and developed a pharmacokinetic-pharmacodynamic (PKPD) model to capture this phenomenon. In vitro static time-kill experiments were performed using polymyxin B at concentrations ranging from 0.125 to 128 mg/L against a clinical A. baumannii isolate at four starting inocula from 105 to 108 CFU/mL. Samples were collected up to 30 h to quantify the viable bacterial burden and were simultaneously modeled in the NONMEM software program. The expression of polymyxin B resistance genes (lpxACD, pmrCAB, and wzc), and genetic modifications were studied by RT-qPCR and DNA sequencing experiments, respectively. The PKPD model included a single homogeneous bacterial population with adaptive resistance. Polymyxin B effect was modeled as a sigmoidal Emax model and the inoculum effect as an increase of polymyxin B EC50 with increasing starting inoculum using a power function. Polymyxin B displayed a reduced activity as the starting inoculum increased: a 20-fold increase of polymyxin B EC50 was observed between the lowest and the highest inoculum. No effects of polymyxin B and inoculum size were observed on the studied genes. The proposed PKPD model successfully described and predicted the pronounced in vitro inoculum effect of A. baumannii on polymyxin B activity. These results should be further validated using other bacteria/antibiotic combinations and in vivo models.

Prevalence and Characterization of the Cefazolin Inoculum Effect in North American Methicillin-Susceptible Staphylococcus aureus Isolates.

Antistaphylococcal penicillins and cefazolin remain the primary treatments for infections with methicillin-susceptible Staphylococcus aureus (MSSA). The cefazolin inoculum effect (CzIE) causes the cefazolin MIC to be elevated in proportion to the number of bacteria in the inoculum. The objective of this multicenter study was to evaluate the prevalence of the CzIE in North American MSSA isolates. Clinical MSSA isolates from six microbiology laboratories in the United States and one microbiology laboratory in Canada were screened for the CzIE by broth microdilution at a standard inoculum (~5 × 105 CFU/mL) and a high inoculum (~5 × 107 CFU/mL). Genome sequencing was performed to further characterize the MSSA isolates. The CzIE was present in 57/305 (18.6%) MSSA isolates, ranging from 0% to 27.9% across study sites. More of the CzIE-positive isolates (29.8%) had standard inoculum cefazolin MICs of 1.0 µg/mL than the CzIE-negative isolates did (3.2%) (P < 0.0001). Conversely, more CzIE-negative isolates (39.5%) had standard inoculum MICs of 0.25 µg/mL than the CzIE positive isolates did (5.3%) (P < 0.0001). The most common BlaZ ß-lactamase types found in the CzIE-positive strains were type C (53.7%) and type A (44.4%). ST8 and ST30 were the most common sequence types among CzIE-positive isolates and correlated with BlaZ type C and A, respectively. The CzIE was present in up to a quarter of clinical MSSA isolates from North American clinical laboratories. Further studies to determine the impact of the presence of the CzIE on clinical outcomes are needed.

Inoculum effect of Enterobacterales co-expressing OXA-48 and CTX-M on the susceptibility to ceftazidime/avibactam and meropenem.

BACKGROUND: The treatment of infections caused by OXA-48/CTX-M-coproducing Enterobacterales may be based on new beta-lactam/beta-lactamase inhibitors, such as ceftazidime/avibactam (CZA), or on high dose of meropenem (MER). However, bacterial density at the infection site may vary widely, and the inoculum effect of such antimicrobial strategies has never been specifically investigated. To determine if CZA or MER susceptibilities are impacted by high inocula of Enterobacterales co-expressing both enzymes: OXA-48 like and CTX-M. METHODS: Determination of an inoculum effect was performed with a standard inoculum of 108 CFU/mL (0.5 McFarland) as recommended by EUCAST guidelines and compared to a twofold increase as well as a tenfold increase (1 McFarland and 5 McFarland respectively). RESULTS: Thirty-nine isolates of ceftazidime-resistant Enterobacterales were included of which 27 (70%) co-expressed OXA-48 + CTX-M-15, 6 (15%) OXA-48 + CTX-M-14, and 6 (15%) OXA-181 + CTX-M-15. The susceptibility to the CZA combination was preserved whatever the inoculum used. Regarding MER, 24 (61.5%) of the isolates were susceptible to MER with the standard inoculum, 19 (48.7%) with a twofold increase, and only 15 (38.5%) with a tenfold increase. CONCLUSION: We showed that in vitro inoculum effect was observed with meropenem but not with CZA for OXA-48- combined with CTX-M-producing Enterobacterales.

A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus.

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-ß-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (107 CFU/ml) was used as a reference standard (cutoff ≥16 µg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates 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 (the 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 isolates from the United States 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.

Assessment of cooperative antibiotic resistance of Salmonella Typhimurium within heterogeneous population.

This study was designed to investigate the cooperative resistance in the mixed culture of antibiotic-sensitive and antibiotic-resistant Salmonella Typhimurium. Strains of S. Typhimurium ATCC 19585 (STS) and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 (STR) grown in single and mixture with 1 × MIC ceftriaxone (CEF) were used to determine the viability, ß-lactamase activity, and gene expression. The MIC50 values of STR to CEF was increased by more than 5-fold with increasing inoculum densities from 102 to 107 CFU/mL. STS was resistant to 1 × MIC CEF in the mixed culture of STS and STR, showing the more than 108 CFU/mL after 20 h of incubation at 37 °C. The highest ß-lactamase activity was 18 µmol/min/mL in the mixed culture, corresponding to the highest relative expression of ß-lactamase-related genes (blaTEM). These results shed new light on the cooperative resistance of antibiotic-sensitive bacteria within a heterogeneous population including ß-lactamase-producing bacteria.

Revisiting the inoculum effect for Streptococcus pyogenes with a hollow fibre infection model.

Severe, invasive Streptococcus pyogenes (Strep A) infections result in greater than 500,000 deaths annually. First line treatment for such infections is benzylpenicillin, often with the addition of clindamycin, but treatment failure can occur with this regimen. This failure has been partially attributed to the inoculum effect, which presents as reduced antibiotic susceptibility during high bacterial density and plateau-phase growth. Hollow fibre infection models (HFIM) have been proposed as an in vitro alternative to in vivo research to study these effects. To re-evaluate the inoculum effect for benzylpenicillin, clindamycin, linezolid, and trimethoprim-sulfamethoxazole using a Strep A HFIM. Differential antibiotic susceptibility of Strep A was measured in a HFIM starting from low- and high-density inocula with an average difference in bacterial concentration of 56-fold. Dynamic antibiotic concentrations were delivered over 48 h to simulate in vivo human pharmacokinetics in an in vitro model. Differences in antibiotic susceptibility were measured by plate count of colony-forming units over time. Inoculum effects were seen in benzylpenicillin and linezolid at 24 h, and benzylpenicillin, linezolid, and clindamycin at 48 h. The effect size was greatest for continuously infused benzylpenicillin at 48 h with a log10-fold difference of 4.02 between groups. No inoculum effect was seen in trimethoprim-sulfamethoxazole, with a maximal log10-fold difference of 0.40. Inoculum effects were seen using benzylpenicillin, linezolid, and clindamycin, which may predict reduced clinical efficacy following treatment delay. The model has proven robust and largely in agreeance with published data, recommending it for further Strep A study.

Novel Insights into the Classification of Staphylococcal ß-Lactamases in Relation to the Cefazolin Inoculum Effect.

Cefazolin has become a prominent therapy for methicillin-susceptible Staphylococcus aureus (MSSA) infections. However, an important concern is the cefazolin inoculum effect (CzIE), a phenomenon mediated by staphylococcal ß-lactamases. Four variants of staphylococcal ß-lactamases have been described based on serological methodologies and limited sequence information. Here, we sought to reassess the classification of staphylococcal ß-lactamases and their correlation with the CzIE. We included a large collection of 690 contemporary bloodstream MSSA isolates recovered from Latin America, a region with a high prevalence of the CzIE. We determined cefazolin MICs at standard and high inoculums by broth microdilution. Whole-genome sequencing was performed to classify the ß-lactamase in each isolate based on the predicted full sequence of BlaZ. We used the classical schemes for ß-lactamase classification and compared it to BlaZ allotypes found in unique sequences using the genomic information. Phylogenetic analyses were performed based on the BlaZ and core-genome sequences. The overall prevalence of the CzIE was 40%. Among 641 genomes, type C was the most predominant ß-lactamase (37%), followed by type A (33%). We found 29 allotypes and 43 different substitutions in BlaZ. A single allotype, designated BlaZ-2, showed a robust and statistically significant association with the CzIE. Two other allotypes (BlaZ-3 and BlaZ-5) were associated with a lack of the CzIE. Three amino acid substitutions (A9V, E112A, and G145E) showed statistically significant association with the CzIE (P = <0.01). CC30 was the predominant clone among isolates displaying the CzIE. Thus, we provide a novel approach to the classification of the staphylococcal ß-lactamases with the potential to more accurately identify MSSA strains exhibiting the CzIE.

Cefazolin Inoculum Effect and Methicillin-Susceptible Staphylococcus aureus Osteoarticular Infections in Children.

Select methicillin-susceptible Staphylococcus aureus (MSSA) strains may produce ß-lactamases with affinity for first-generation cephalosporins (1GCs). In the setting of a high inoculum, these ß-lactamases may promote the cleavage of 1GCs, a phenomenon known as the cefazolin inoculum effect (CzIE). We evaluated the prevalence and impact of CzIE on clinical outcomes among MSSA acute hematogenous osteomyelitis (AHO) cases. MSSA AHO isolates obtained from two children's hospitals between January 2011 and December 2018 were procured through ongoing surveillance studies. Isolates were tested for CzIE via a broth macrodilution assay using an inoculum of 107 CFU/ml; CzIE was defined as a cefazolin MIC of ≥16 µg/ml. Isolates were characterized by accessory gene regulator group (agr). The progression from acute to chronic osteomyelitis was considered an important outcome. A total of 250 cases with viable isolates were included. Notably, 14.4% of isolates exhibited CzIE with no observed temporal trend; and 4% and 76% of patients received a 1GC as an empirical and definitive therapy, respectively. CzIE isolates were more often resistant to clindamycin, belonged to agrIII, and associated with the development of chronic osteomyelitis. In multivariable analyses, agrIII, multiple surgical debridements, delayed source control, and CzIE were independently associated with progression to chronic osteomyelitis. A higher rate of chronic osteomyelitis was observed with CzIE isolates regardless of definitive antibiotic choice. CzIE is exhibited by 14.4% of MSSA AHO isolates in children. CzIE is independently associated with progression to chronic osteomyelitis in cases of AHO irrespective of final antibiotic choice. These data suggest that negative outcomes reported with CzIE may more accurately reflect strain-dependent virulence factors rather than true antibiotic failure.

Inoculum effect of methicillin-susceptible Staphylococcus aureus against broad-spectrum beta-lactam antibiotics.

Scarce information concerning the inoculum effect (InE) of methicillin-susceptible Staphylococcus aureus (MSSA) against broad-spectrum ß-lactam antibiotics is available. We investigated the InE of MSSA against ceftriaxone, cefepime, meropenem, ampicillin/sulbactam and piperacillin/tazobactam. The bacteraemic MSSA isolates were collected at ten Korean general hospitals from Sep 2013 to Mar 2015. The InE was defined if MICs of antibiotics at high inoculum (HI, ~5 × 107 CFU/ml) increased beyond the susceptible range compared to those at standard inoculum (SI, ~5 × 105 CFU/ml). All isolates were sequenced for blaZ gene typing. Among 302 MSSA isolates, 254 (84.1%) were positive for blaZ; types A, B, C and D were 13.6%, 26.8%, 43.4% and 0.3%, respectively. Mean HI MICs of all tested antibiotics were significantly increased and increases in HI MIC of piperacillin/tazobactam (HI, 48.14 ± 4.08 vs. SI, 2.04 ± 0.08 mg/L, p < 0.001) and ampicillin/sulbactam (HI, 24.15 ± 1.27 vs. SI, 2.79 ± 0.11 mg/L, p < 0.001) were most prominent. No MSSA isolates exhibited meropenem InE, and few isolates exhibited cefepime (0.3%) and ceftriaxone (2.3%) InE, whereas 43.0% and 65.9% of MSSA isolates exhibited piperacillin/tazobactam and ampicillin/sulbactam InE, respectively. About 93% of type C blaZ versus 45% of non-type C exhibited ampicillin/sulbactam InE (p < 0.001) and 88% of type C blaZ versus 9% of non-type C exhibited piperacillin/tazobactam InE (p < 0.001). A large proportion of MSSA clinical isolates, especially those positive for type C blaZ, showed marked ampicillin/sulbactam InE and piperacillin/tazobactam.

Adjunctive Clavulanic Acid Abolishes the Cefazolin Inoculum Effect in an Experimental Rat Model of Methicillin-Sensitive Staphylococcus aureus Endocarditis.

We tested the ability of clavulanic acid to restore the efficacy of cefazolin against Staphylococcus aureus TX0117, which exhibits the cefazolin inoculum effect (CzIE). In the rat infective endocarditis model, the coadministration of cefazolin plus clavulanic acid resulted in a significant reduction of bacterial counts (7.1 ± 0.5 log10 CFU/g) compared to that with cefazolin alone (2 ± 0.6 log10 CFU/g; P < 0.0001). The addition of a ß-lactamase inhibitor may be a viable strategy for overcoming the CzIE.

Prevalence of a Cefazolin Inoculum Effect Associated with blaZ Gene Types among Methicillin-Susceptible Staphylococcus aureus Isolates from Four Major Medical Centers in Chicago.

The efficacy of cefazolin with high-inoculum methicillin-susceptible Staphylococcus 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 staphylococcal blaZ gene 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 × 105 CFU/ml) and a high inoculum (HI; 5 × 107 CFU/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 the blaZ gene, 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%) were blaZ positive, with the blaZ types led by gene type C (40%). CInE was seen in 45 blaZ-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 A blaZ gene from MSSA isolates in Chicago is low. Our predilection for cefazolin use, even early in the management of hospitalized MSSA infections, is tenable.

The Inoculum Effect in the Era of Multidrug Resistance: Minor Differences in Inoculum Have Dramatic Effect on MIC Determination.

The observed MIC may depend on the number of bacteria initially inoculated into the assay. This phenomenon is termed the inoculum effect (IE) and is often most pronounced for ß-lactams in strains expressing ß-lactamase enzymes. The Clinical and Laboratory Standards Institute (CLSI)-recommended inoculum is 5 × 105 CFU ml-1 with an acceptable range of 2 × 105 to 8 × 105 CFU ml-1 IE testing is typically performed using an inoculum 100-fold greater than the CLSI-recommended inoculum. Therefore, it remains unknown whether the IE influences MICs during testing performed according to CLSI guidelines. Here, we utilized inkjet printing technology to test the IE on cefepime, meropenem, and ceftazidime-avibactam. First, we determined that the inkjet dispense volume correlated well with the number of bacteria delivered to microwells in 2-fold (R2 = 0.99) or 1.1-fold (R2 = 0.98) serial dilutions. We then quantified the IE by dispensing orthogonal titrations of bacterial cells and antibiotics. For cefepime-resistant and susceptible dose-dependent strains, a 2-fold increase in inoculum resulted in a 1.6 log2-fold increase in MIC. For carbapenemase-producing strains, each 2-fold reduction in inoculum resulted in a 1.26 log2-fold reduction in meropenem MIC. At the lower end of the CLSI-allowable inoculum range, minor error rates of 34.8% were observed for meropenem when testing a resistant-strain set. Ceftazidime-avibactam was not subject to an appreciable IE. Our results suggest that IE is sufficiently pronounced for meropenem and cefepime in multidrug-resistant Gram-negative pathogens to affect categorical interpretations during standard laboratory testing.

Inoculum effect of high concentrations of methicillin-susceptible Staphylococcus aureus on the efficacy of cefazolin and other beta-lactams.

The existence of a cefazolin inoculum effect (InE) of methicillin-susceptible Staphylococcus aureus (MSSA), which is speculated to be a reason for cefazolin treatment failure in MSSA infections, is controversial. In Japan, although cefazolin is one of the therapeutic choices for patients with MSSA infection, there are few reports of this effect. Additionally, the association between InE and blaZ type in beta-lactams other than cefazolin has not been well documented. In this study, we confirmed an MSSA InE in several beta-lactams, including cefazolin, and its relationship with blaZ, using 52 MSSA isolates from blood cultures. Three isolates (5.8%) that possessed type A blaZ showed a pronounced cefazolin InE. Five isolates (9.6%) showed pronounced InE with sulbactam/ampicillin; four isolates had type C blaZ and one had type A blaZ. However, we confirmed InE in MSSA isolates with blaZ not only type A and C but also B and D. For cefotaxime, ceftriaxone, imipenem, and meropenem, regardless of the presence of blaZ, we did not observe a significant increase in MICs at a high inoculum of MSSA. Hence, our results suggest that the above four beta-lactams are good alternatives to cefazolin if InE leads to treatment failure in a patient.

Antimicrobial Effects of ß-Lactams on Imipenem-Resistant Ceftazidime-Susceptible Pseudomonas aeruginosa.

We studied the resistance mechanism and antimicrobial effects of ß-lactams on imipenem-resistant Pseudomonas aeruginosa isolates that were susceptible to ceftazidime as detected by time-kill curve methods. Among 215 P. aeruginosa isolates from hospitalized patients in eight hospitals in the Republic of Korea, 18 isolates (23.4% of 77 imipenem-resistant isolates) were imipenem resistant and ceftazidime susceptible. Multilocus sequence typing revealed diverse genotypes, which indicated independent emergence. These 18 isolates were negative for carbapenemase genes. All 18 imipenem-resistant ceftazidime-susceptible isolates showed decreased mRNA expression of oprD, and overexpression of mexB was observed in 13 isolates. In contrast, overexpression of ampC, mexD, mexF, or mexY was rarely found. Time-kill curve methods were applied to three selected imipenem-resistant ceftazidime-susceptible isolates at a standard inoculum (5 × 105 CFU/ml) or at a high inoculum (5 × 107 CFU/ml) to evaluate the antimicrobial effects of ß-lactams. Inoculum effects were detected for all three ß-lactam antibiotics, ceftazidime, cefepime, and piperacillin-tazobactam, against all three isolates. The antibiotics had significant killing effects in the standard inoculum, but no effects in the high inoculum were observed. Our results suggest that ß-lactam antibiotics should be used with caution in patients with imipenem-resistant ceftazidime-susceptible P. aeruginosa infection, especially in high-inoculum infections such as endocarditis and osteomyelitis.