Single-strain regression analysis evaluating disc potencies of flumequine and enrofloxacin for testing Aeromonas sobria and Vibrio anguillarum.

Two quite different disc contents are used for antimicrobial susceptibility testing of two fluoroquinolone drugs, flumequine and enrofloxacin, in the disc diffusion test, 30 and 5 µg, respectively. Using the SRA method, single-strain regression analysis, we studied the impact of disc content when testing two relevant bacterial species, Aeromonas sobria and Vibrio anguillarum. There were no major differences between the antimicrobial regression lines for the two species. Wild-type strains produced acceptable zones of inhibition over a wide range of disc contents. The flumequine 30 µg disc should be lowered in its drug content. No rational reasons for choosing so different disc contents for the two antimicrobials were apparent. At present, the choice of disc content for new antimicrobials are outside the realm of clinical microbiologists. It is recommended that reference authorities, such as EUCAST, CLSI and USCAST, are consulted for the choice of disc contents in the future.

Antimicrobial resistance surveillance in Escherichia coli by using normalized resistance interpretation.

OBJECTIVES: To improve antimicrobial surveillance accuracy for results obtained by disk diffusion for porcine Escherichia coli, by comparing traditional clinical breakpoint interpretation with the Normalized Resistance Interpretation (NRI) method. METHODS: The susceptibilities of 921 E. coli isolates from clinically healthy pigs at slaughter age was determined for 15 antimicrobials by the Kirby Bauer disk diffusion technique. NRI with previously established optimal controlled parameters for E. coli ATCC25922 was used to reconstruct the fully susceptible population of the tested E. coli isolates. Based on a lower limit for susceptibility, set at 2.5 standard deviations below the mean of the reconstructed susceptible population, the non-wild type percentage isolates was compared with the clinical resistance percentage. RESULTS: The NRI method was applicable for 11 out of the 15 antimicrobials tested. Antimicrobials for which no normal distribution of inhibition zones for the population of susceptible isolates was seen, could not be used to reconstruct the susceptible population. Clinical breakpoints much lower than the epidemiological cut-off values resulted into presumptively identifying isolates as clinically susceptible, but likely carrying acquired resistance determinants. Otherwise, clinical breakpoints did cut through the WT population for several antibiotics tested, categorizing isolates from the WT population as not susceptible. CONCLUSIONS: NRI was shown to be a valid method to define the WT population for disk diffusion outcomes, provided a normal distribution of the susceptible bacterial species population is present. Until international harmonization of breakpoints is achieved, it might give rise to a wide application in monitoring antimicrobial resistance in veterinary medicine.

Normalized resistance interpretation, the NRI method: Review of NRI disc test applications and guide to calculations.

The normalized resistance interpretation (NRI) method was developed in response to a call for a method to calibrate disc diffusion test results making inter-laboratory comparisons possible. The main use of NRI so far has been in individual laboratories, in medical and veterinary medicine and in the field of marine microbiology. The applications of NRI for disc diffusion tests are reviewed and, in addition, a detailed description of the calculation procedure is presented. NRI provides a fully objective method for ECOFF calculations of disc diffusion antimicrobial susceptibility test results.

Assessment of antibiotic resistance of Escherichia coli isolates and screening of Salmonella spp. in wild ungulates from Portugal.

Antibiotic resistance is an emerging global problem. Wild animals are rarely exposed to antibiotics and therefore low levels of antibiotic resistance are expected. However, the growing interactions of these animals with humans and livestock may have a huge impact on their bacterial flora. This study aimed to assess the levels of antibiotic resistance in Escherichia coli isolated from widespread wild ungulates in Portugal. The interpretation of inhibition zone diameters was performed according to clinical breakpoints and epidemiological cut-offs, determined with the normalized resistance interpretation (NRI) method. For clinical breakpoints, 16% of the isolates were resistant to at least one antibiotic, including ampicillin (10%), tetracycline (9%), streptomycin (5%) co-trimoxazole (4%), amoxicillin/clavulanic acid (1%) and cefoxitin (1%). The levels of resistance detected in E. coli strains isolated from wild boar were statistically different for ampicillin and co-trimoxasol. According to NRI cut-offs, 10% of the population showed a non-wild-type phenotype against at least one antibiotic, also including tetracycline (9%), co-trimoxazole (6%), streptomycin (4%), ampicillin (2%) and amoxicillin/clavulanic acid (1%). Considering this parameter of comparison, no statistically different levels of resistance were identified between E. coli recovered from the three wild ungulates. Screening of Salmonella spp., which can be potentially pathogenic, was also performed, revealing that its prevalence was very low (1.5%). The study demonstrated that wild ungulates from Portugal are also reservoirs of antibiotic-resistant bacteria.

Estimating the precision of disc diffusion antibiotic susceptibility data published by the European committee on antimicrobial susceptibility testing.

Analysis of 163 disc diffusion data sets, 115 for bacterial species groups and 48 for types strains, published by EUCAST, was used to optimize the setting of the parameters of a standardized protocol for normalized resistance interpretation of these data. The standard deviations of the normalized distributions of these data sets, calculated using this standardized protocol, were shown to be independent of the means of their respective distributions. These standard deviations could, therefore, be used as a metric to quantify the precision of disc diffusion data sets. The median value of the standard deviations for 115 EUCAST data sets produced in multiple laboratory studies of bacterial species groups was 2.3 mm and that for the 48 EUCAST data sets produced in multiple laboratory studies of type strains was 1.5 mm. It is argued that this standardized method for estimating the precision of disc diffusion data provides a tool by which individual laboratories can assess the quality of the disc diffusion data they produce.

Vibrio ordalii antimicrobial susceptibility testing--modified culture conditions required and laboratory-specific epidemiological cut-off values.

The present study aimed to determine oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXO) MICs and zone diameters for 24 Chilean Vibrio ordalii isolates using the methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals and the methods for antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals guidelines published by the Clinical Laboratory Standards Institute (CLSI). The results were then used in a normalized resistance interpretation (NRI) analysis to establish tentative laboratory-specific epidemiological cut-off (ECOFF) values. MIC results were similar at the two tested temperatures (22 °C and 18 °C). At 18 °C, the NRI analysis of OTC, FLO and OXO MIC data calculated laboratory-specific ECOFF values and non-wild-type (NWT) rates to be ≤4 mg/l (24%), ≤16 mg/l (4%) and ≤8 mg/l (25%), respectively. Tests performed with all V. ordalii isolates following the officially recommended incubation temperature (22 °C) revealed difficulties in measuring inhibition zone diameters. When disk diffusion tests were performed using Mueller-Hinton agar with 1% NaCl (MHA-1) at 18 °C the inhibition zone diameter distributions showed the formation of WT populations which could be defined using NRI analysis. For OTC the laboratory-specific ECOFF value was ≥38 mm with NWT rate of 16.7%. For FLO and OXO, the laboratory-specific ECOFF values were ≥38 and ≥40, respectively, generating NWT rates of 25 and 46%, respectively. Although the CLSI suggests testing Vibrio spp. on MHA-1 at 22 °C, we found measurements of the 24 isolates were better defined and normally distributed at 18 °C. This is the first study determining the MIC and disk diffusion test of V. ordalii isolated from diseased salmonids, where laboratory-specific ECOFF values could be established. Also resistance to OTC, FLO and OXO among some Chilean isolates was demonstrated.

Increase of ß-lactam-resistant invasive Haemophilus influenzae in Sweden, 1997 to 2010.

The proportions of Haemophilus influenzae resistant to ampicillin and other ß-lactam antibiotics have been low in Sweden compared to other countries in the Western world. However, a near-doubled proportion of nasopharyngeal Swedish H. influenzae isolates with resistance to ß-lactams has been observed in the last decade. In the present study, the epidemiology and mechanisms of antimicrobial resistance of H. influenzae isolates from blood and cerebrospinal fluid in southern Sweden from 1997 to 2010 (n = 465) were studied. Antimicrobial susceptibility testing was performed using disk diffusion, and isolates with resistance to any tested ß-lactam were further analyzed in detail. We identified a significantly increased (P = 0.03) proportion of ß-lactam-resistant invasive H. influenzae during the study period, which was mainly attributed to a significant recent increase of ß-lactamase-negative ß-lactam-resistant isolates (P = 0.04). Furthermore, invasive ß-lactamase-negative ß-lactam-resistant H. influenzae isolates from 2007 and onwards were found in higher proportions than the corresponding proportions of nasopharyngeal isolates in a national survey. Multiple-locus sequence typing (MLST) of this group of isolates did not completely separate isolates with different resistance phenotypes. However, one cluster of ß-lactamase-negative ampicillin-resistant (BLNAR) isolates was identified, and it included isolates from all geographical areas. A truncated variant of a ß-lactamase gene with a promoter deletion, bla(TEM-1)-PΔ dominated among the ß-lactamase-positive H. influenzae isolates. Our results show that the proportions of ß-lactam-resistant invasive H. influenzae have increased in Sweden in the last decade.

Throat carriage rate and antimicrobial susceptibility pattern of group A Streptococci (GAS) in healthy Ethiopian school children.

BACKGROUND: Group A Streptococci (GAS) or Streptococcus pyogenes are the most frequent cause of pharyngitis and skin infection in children and lead to post infection complications including acute rheumatic fever and glomerulonephritis. Pharyngeal carriage rates of GAS among healthy school children vary with geographical location and seasons. There is not much information on the screening of children for carriage of GAS in Ethiopia. OBJECTIVES: The study aimed at assessing the carriage rate of Group A Streptococci and antimicrobial susceptibility of the isolates in healthy Ethiopian school children. METHODS: A total of 937 children residing in Addis Ababa (n=491), Gondar (n=265) and Dire-Dawa (n=181) were investigated during a period between November 2004 and January 2005. Throat specimens were collected and cultured using standard procedure. Beta haemolytic streptococci were serogrouped by agglutination tests using specific antisera. Antimicrobial susceptibility testing of the isolates was performed by diffusion method. RESULTS: The median and the mean ages of the study participants were 11 (range 6-14) years. Girls constituted 52% (486/937) of the study participants. A total of 167 (17.8%) beta haemolytic streptococci were recovered from 937 children investigated GAS accounted for 91/167 (54.5%) of beta hemolytic streptococcal isolates. The carrier rate for GAS was 9.7% (91/937) of the screened children followed by group G with 3.2% (30/937) and group C streptococci with 2.2% (21/937). All GAS isolates were sensitive to oxacillin, penicillin, erythromycin, clindamycin and trimethoprim-sulphamethoxazole. Lower frequency of resistance was observed against tetracycline and vanocmycin. CONCLUSION: The present study revealed that GAS was the most predominant beta-haemolytic streptococcus among healthy Ethiopian school children. Our results showed that pharyngeal carriage of GAS in school children should not be underestimated. Therefore it is recommended to conduct regular screening and GAS surveillance in schools, and maintain rational use of antibiotics to minimize GAS resistance.

Setting interpretive breakpoints for antimicrobial susceptibility testing using disk diffusion.

Antimicrobial susceptibility testing plays a key role in clinical microbiology. The disk diffusion test dates back to the 1940s and became standardised from the 1950s, with the International Collaborative Study (ICS) and National Committee for Clinical Laboratory Standards (NCCLS) as the two major standards. Interlaboratory variation of disk test results was recognised early but has never been dealt with in a satisfactory manner. The error-rate bounded method was described in 1974 and its role is discussed. Species-specific susceptibility interpretation was coined in 1980 for Proteus mirabilis and chloramphenicol. In the late 1970s, more extensive use of species-specific breakpoints was introduced in Lund (Sweden). At the same time, P. Mouton constructed species-specific regression lines and pointed out the difficulties with narrow ranges of minimal inhibitory concentration (MIC) values. A more general use of species-specific regression lines was made possible with single-strain regression analysis, using one well-defined strain tested in disk diffusion with a range of disk contents. This method made it possible to calibrate the disk test in an individual laboratory. Other methods to achieve such calibration are also described. A recent method, 'MIC-coloured zone diameter histogram-technique', has proven useful for the validation of species-specific interpretive breakpoints. The microbiological breakpoint proposed by Williams in 1990 has experienced a renaissance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off value (ECOFF). MIC and zone diameter distributions with accompanying ECOFFs for species-antimicrobial combinations are published on the EUCAST website. A method for the reconstruction of wild-type zone diameter populations, namely normalised resistance interpretation, is described. This method can produce resistance figures that are truly comparable between laboratories.

Normalized resistance interpretation as a tool for establishing epidemiological MIC susceptibility breakpoints.

Normalized resistance interpretation (NRI) utilizes the fact that the wild-type population on the sensitive side is not affected by resistance development, and therefore a normalized reconstruction of the peak can be performed. The method was modified for MIC distributions by the introduction of helper variables, in-between values assigned the mean of the neighboring numbers of isolates. This method was used on Staphylo- coccus aureus and Escherichia coli MIC distributions for 27 antimicrobials each and obtained from the EUCAST (European Committee on Antimicrobial Susceptibility Testing) website (http://www.eucast.org/mic_distributions/). The number of isolates in each of the 54 distributions ranged from 40 to 124,472. NRI produced normalized distributions in all cases. Cutoff values were calculated for +2.0 and +2.5 standard deviations (SD) above the means and then rounded up to nearest regular MIC dilution step. EUCAST also show cutoff values, ECOFF values, which were used as the reference. The NRI generated +2.0 SD values showed the best agreement with 26 of 27 within ±1 dilution step and 17 exactly on the ECOFF values for Staphylococcus aureus, and 25 of 27 within ±1 dilution step and 14 right on the ECOFF values for Escherichia coli. NRI offers an objective method for the reconstruction of the wild-type population in an MIC distribution for a given bacterial species and an antimicrobial agent. This method offers a new tool in comparative susceptibility studies such as global surveillance of resistance, as well as in quality control in individual laboratories.

Antimicrobial resistance 1979-2009 at Karolinska hospital, Sweden: normalized resistance interpretation during a 30-year follow-up on Staphylococcus aureus and Escherichia coli resistance development.

To utilize a material of inhibition zone diameter measurements from disc diffusion susceptibility tests between 1979 and 2009, an objective setting of epidemiological breakpoints was necessary because of methodological changes. Normalized resistance interpretation (NRI) met this need and was applied to zone diameter histograms for Staphylococcus aureus and Escherichia coli isolates. The results confirmed a slow resistance development as seen in Northern countries. The S. aureus resistance levels for erythromycin, clindamycin and fusidic acid in 2009 were 3.2%, 1.8% and 1.4% with denominator correction. A rise in resistance to four antimicrobials in 1983 was probably because of a spread of resistant Methicillin Susceptible Staphylococcus Aureus (MSSA). For E. coli, the denominator-corrected resistance levels in 2009 were 27% for ampicillin, around 3% for third-generation cephalosporins, 0.1% for imipenem, 2.5% for gentamicin, 19% for trimethoprim, 4.5% for co-trimoxazole, 1.2% for nitrofurantoin and 9% for ciprofloxacin. The temporal trends showed a rise in fluoroquinolone resistance from 1993, a parallel increase in gentamicin resistance, a substantial increase in trimethoprim and sulphonamide resistance in spite of decreased consumption, and a steady rise in ampicillin resistance from a constant level before 1989. A short review of global resistance surveillance studies is included.

Antimicrobial activity of tigecycline and comparative agents against clinical isolates of staphylococci and enterococci from ICUs and general hospital wards at three Swedish university hospitals.

The activities of tigecycline and comparative agents on staphylococci and enterococci isolated from patients at general hospital wards (GHWs) and intensive care units (ICUs) at 3 university hospitals in Sweden were investigated. Oxacillin disc diffusion and minimal inhibitory concentration with E-test were used. The presence of mecA, vanA or vanB genes was determined with PCR. Statistically significant higher incidence of clindamycin, fusidic acid, rifampicin and multidrug-resistant CoNS was found at ICUs compared to GHWs. Resistance rates were low among S. aureus. Tigecycline, linezolid and vancomycin were the only agents with high activity against methicillin-resistant S. aureus and multidrug-resistant CoNS. Resistance rates were low among E. faecalis, except for high-level gentamicin-resistant (HLGR) E. faecalis. E. faecium showed high resistance rates to ampicillin, piperacillin/tazobactam and imipenem. The HLGR rates among E. faecium were lower than the rates for E. faecalis. Tigecycline and linezolid were the only drugs with high activity against all enterococci including vancomycin-resistant enterococci. No statistically significant differences in susceptibility rates were found between the ward levels for S. aureus and enterococcal isolates and no statistically significant differences were found between the hospitals.

Rapid dissemination and diversity of CTX-M extended-spectrum beta-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America.

A survey carried out in 2005 among members of a healthy population of children living in Bolivia and Peru revealed that fecal carriage of Escherichia coli strains resistant to expanded-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002 (1.7% in 2005 versus 0.1% in 2002). In this work, we demonstrated that this phenomenon was mainly related to the dissemination of CTX-M-type extended-spectrum beta-lactamase (ESBL) determinants among commensal E. coli strains. Of 50 ESBL-producing isolates collected in the 2005 survey, 44 harbored a CTX-M-type and 6 an SHV-type (SHV-2 or SHV-12) ESBL. Compared to 2002 results, an increased diversity of CTX-M-type ESBLs was also observed: members of the CTX-M-1 group (CTX-M-15) emerged in Bolivia (where only CTX-M-2 was observed in 2002), while members of the CTX-M-9 group (CTX-M-14 and CTX-M-24) emerged in Peru (where only CTX-M-15 and CTX-M-2 were observed in 2002). A new CTX-M-2 variant named CTX-M-56 was also detected. Molecular characterization of the CTX-M-producing isolates and gene transfer experiments suggested that different mechanisms could be involved in the spreading of different CTX-M group determinants and revealed that additional resistance determinants for non-beta-lactam antibiotics were preferentially carried by plasmids encoding certain CTX-M variants (CTX-M-15 and variants of the CTX-M-2 group). Three CTX-M-15-encoding conjugative plasmids from Peruvian isolates carried the new fluoroquinolone resistance gene aac(6')-Ib-cr. To our best knowledge, this is the first report of the detection of aac(6')-Ib-cr in Latin America.