Evaluation of XGEN Multi Sepsis Flow Chip Molecular Assay for Early Diagnosis of Bloodstream Infection.

Among healthcare-associated infections that can affect a critically ill patient, bloodstream infections are one of the most frequent causes of mortality, especially in hospitalized patients. The objective of this work is to evaluate the performance of the XGEN Multi Sepsis Flow Chip for the rapid diagnosis of bloodstream infections compared with conventional tests. In total, 101 positive blood culture samples were included, and the results obtained by the phenotypic conventional method (culture with susceptibility profile) were compared with results obtained by the XGEN Multi Sepsis Flow Chip. This molecular assay allows the simultaneous detection of the main bloodstream infection pathogens, and their most common antibiotic resistance markers in a short period of time. It was possible to observe substantial agreement between the methods for identifying the genus of pathogens. Considering species, the agreement was excellent. In relation to susceptibility, excellent agreement was noted between the detected resistance genes and susceptibility profile obtained through conventional antibiograms. The evaluated assay presented very early and satisfactory results for identification and detection of resistance genes of the main pathogens involved in bloodstream infections.

Co-carriage of Plasmid NDM and Chromosomal KPC in Klebsiella pneumoniae ST255 Human Wound Isolate in Brazil.

Multidrug-resistant K. pneumoniae is one of the main causes of hospital-acquired infections worldwide and frequently carries antimicrobial resistance genes in moving elements. In this study, we described a K. pneumoniae clinical isolate carrying simultaneous chromosomal blaKPC, and plasmid-mediated blaNDM and blaOXA-9. The isolate is multidrug-resistant and belongs to ST 225. While blaKPC were identified in the chromosome, the blaNDM was mediated by IncFII(K) plasmid and the blaOXA-9, in a IncFIB(K) plasmid. The blaKPC context was composed by Tn4401 transposon and two insertion sequences ISKpn6 and ISKpn7. The co-production of diverse ß-lactamases brings an alert about a new adaptive profile of K. pneumoniae strains and their dissemination in the hospital-acquired infectious.

Genomic analyses of ciprofloxacin-resistant Neisseria gonorrhoeae isolates recovered from the largest South American metropolitan area.

We sequenced 13 Neisseria gonorrhoeae isolates exhibiting distinct susceptibility profiles and which were recovered over 12 years in the metropolitan region of São Paulo, Brazil. Whole Genome Sequencing (WGS) was performed on an Illumina MiSeq™ 2 × 300 bp paired-end reads. Bioinformatics analyses were carried out using CGE, PATRIC, and BLAST databases for manual curation of obtained genomes. Multilocus sequence typing (MLST) analysis identified seven STs, namely ST1580, ST1590, ST1901, ST1902, ST8161, ST9363, and ST15640. Moreover, a diversity of mutations was observed in MtrR/G45D-A39T, PIB/G120K-A121S, and PBP1/L421P. Mutations associated with sulfonamides (DHPS/R228S) and rifampicin (RNAP/H552N) were also detected, as well as tetracycline resistance determinants, namely rpsJ/V57M and tet(M). The results presented herein can contribute to the knowledge of N. gonorrhoeae strains circulating in Sao Paulo, Brazil.

In vitro synergy of ceftolozane/tazobactam in combination with fosfomycin or aztreonam against MDR Pseudomonas aeruginosa.

OBJECTIVES: Carbapenem-resistant Pseudomonas aeruginosa (CR-PSA) imposes great limitations on empirical therapeutic choices, which are further complicated by metallo-ß-lactamase production. This study evaluated in vitro antimicrobial synergy of ceftolozane/tazobactam in combination with aztreonam and fosfomycin against MDR PSA. METHODS: MICs were determined by broth microdilution and gradient strips. The effect of ceftolozane/tazobactam+aztreonam and ceftolozane/tazobactam+fosfomycin combinations were tested against 27 MDR PSA isolates carrying blaSPM-1 (n = 13), blaIMP (n = 4), blaVIM (n = 3), blaGES-1 (n = 2) and blaCTX-M-like (n = 2), and 3 isolates with no acquired ß-lactamase production detected by gradient diffusion strip crossing (GDSC). Six genetically unrelated SPM-1-producing isolates were also evaluated by time-kill analysis (TKA). RESULTS: All CR-PSA isolates harbouring blaSPM-1, blaGES-1 and blaIMP-1 were categorized as resistant to ceftolozane/tazobactam, meropenem and fosfomycin, with 70% being susceptible to aztreonam. Synergism for ceftolozane/tazobactam+fosfomycin and ceftolozane/tazobactam+aztreonam combinations was observed for 88.9% (24/27) and 18.5% (5/27) of the isolates by GDSC, respectively. A 3- to 9-fold reduction in ceftolozane/tazobactam MICs was observed, depending on the combination. Ceftolozane/tazobactam+fosfomycin was synergistic by TKA against one of six SPM-1-producing isolates, with additional non-synergistic bacterial density reduction for another isolate. Aztreonam peak concentrations alone demonstrated a ≥3 log10 cfu/mL reduction against all six isolates, but all strains were within the susceptible range for the drug. No antagonism was observed. CONCLUSIONS: In the context of increasing CR-PSA and the genetic diversity of resistance mechanisms, new combinations and stewardship strategies may need to be explored in the face of increasingly difficult to treat pathogens.

Gut Microbiota Differences in Children From Distinct Socioeconomic Levels Living in the Same Urban Area in Brazil.

OBJECTIVE: To compare gut microbiota in impoverished children versus children of high socioeconomic status living in the same urban area in Brazil. METHODS: A cross-sectional study was conducted to evaluate 100 children living in a slum and 30 children from a private school, ages between 5 and 11 years old, in Sao Paulo State, Brazil. To characterize the groups, data based on socioeconomic status, sanitation, and housing conditions were collected. Anthropometric measurements and neonatal data were obtained from both groups. Gut microbiota were quantified in fecal samples by real-time polymerase chain reaction. RESULTS: The children in the private school group had higher rates of cesarean delivery and premature birth than the children in the slum group. Staphylococcus aureus (90% vs 48.0%) and Clostridium difficile (100% vs 43.0%) were more commonly found in the children from the private school than in the impoverished children (P < 0.0001). C perfringens was most frequently identified in the group of children from the slum (92.0% vs 80%; P = 0.064). Higher counts of total eubacteria, Firmicutes and Bacteroidetes phyla organisms, Escherichia coli, Lactobacillus spp., and Methanobrevibacter smithii were found in the children living in poverty, whereas higher counts of Salmonella spp., C difficile, and C perfringens were observed in the children living in satisfactory housing conditions (P < 0.05). CONCLUSIONS: Important differences were observed between the gut microbiota of children living under distinct socioeconomic and environmental conditions within the same city. Our findings suggest that children of high socioeconomic status have less favorable gut microbiota than do children who live in poverty.

Characterization of ocular methicillin-resistant Staphylococcus epidermidis isolates belonging predominantly to clonal complex 2 subcluster II.

Staphylococcus epidermidis is an abundant member of the microbiota of the human skin and wet mucosa, which is commonly associated with sight-threatening infections in eyes with predisposing factors. Ocular S. epidermidis has become notorious because of its capability to form biofilms on different ocular devices and due to the evolving rates of antimicrobial resistance. In this study, the molecular epidemiology of 30 ocular methicillin-resistant S. epidermidis (MRSE) isolates was assessed using multilocus sequence typing (MLST). Antimicrobial resistance, accessory gene-regulator and staphylococcal cassette chromosome mec (SCCmec) types, biofilm formation, and the occurrence of biofilm-associated genes were correlated with MLST clonal complexes. Sequence types (STs) frequently found in the hospital setting were rarely found in our collection. Overall, 12 different STs were detected with a predominance of ST59 (30%), ST5 and ST6 (13.3% each). Most of the isolates (93.3%) belonged to the clonal complex 2 (CC2) and grouped mainly within subcluster CC2-II (92.9%). Isolates grouped within this subcluster were frequently biofilm producers (92.3%) with a higher occurrence of the aap (84.5%) and bhp (46.1%) genes compared to icaA (19.2%). SCCmec type IV (53.8%) was predominant within CC2-II strains, while 38.4% were nontypeable. In addition, CC2-II strains were frequently multidrug resistant (80.7%) and demonstrated to be particularly resistant to ciprofloxacin (80.8%), ofloxacin (77%), azithromycin (61.5%), and gentamicin (57.7%). Our findings demonstrate the predominance of a particular MRSE cluster causing ocular infections, which was associated with high rates of antimicrobial resistance and particularly the carriage of biofilm-related genes coding for proteinaceous factors implicated in biofilm accumulation.

Rapid detection of carbapenemase genes by multiplex real-time PCR.

OBJECTIVES: To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). METHODS: A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). RESULTS: Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. CONCLUSIONS: The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

Broad-spectrum antimicrobial consumption trends and correlation with bacterial infections and antimicrobial resistance over 5 years.

OBJECTIVES: Consumption trends of four broad-spectrum antimicrobials and their correlation with antimicrobial resistance in Gram-negative bacilli (GNB) from 2013-2017 within intensive care units (ICUs) were explored. METHODS: Consumption of meropenem (MEM), polymyxin B (PMB), piperacillin/tazobactam (TZP) and cefepime (FEP) in defined daily doses per 1000 patient-days (DDD/1000PD) was measured. Infection-related GNB isolates were grouped according to specific resistance profiles. Time series of antimicrobial consumption and their parametric correlation with each grouped resistant GNB were explored. RESULTS: A total of 1423 GNB were evaluated. A significant linear decline in consumption was observed for MEM [slope -3.88, 95% confidence interval (CI) -4.96 to -2.81; P < 0.0001] and PMB (slope -3.51, 95% CI -5.528 to -1.495; P = 0.0009). A significant decline in MEM-non-susceptible Acinetobacter spp. (R2 = 0.476; P = 0.006) and an increase in FEP-non-susceptible Escherichia coli (R2 = 0.124; P = 0.006) was observed. A significant correlation between MEM consumption and MEM-non-susceptible Acinetobacter spp. (r = 0.43; P = 0.001) was observed. MEM consumption and MEM-non-susceptible Acinetobacter spp. showed a positive correlation. CONCLUSION: Reduction in the consumption of broad-spectrum antimicrobials may alter the frequency of infection-related isolates and their antimicrobial resistance profiles.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

A spatial approach for the epidemiology of antibiotic use and resistance in community-based studies: the emergence of urban clusters of Escherichia coli quinolone resistance in Sao Paulo, Brasil.

BACKGROUND: Population antimicrobial use may influence resistance emergence. Resistance is an ecological phenomenon due to potential transmissibility. We investigated spatial and temporal patterns of ciprofloxacin (CIP) population consumption related to E. coli resistance emergence and dissemination in a major Brazilian city. A total of 4,372 urinary tract infection E. coli cases, with 723 CIP resistant, were identified in 2002 from two outpatient centres. Cases were address geocoded in a digital map. Raw CIP consumption data was transformed into usage density in DDDs by CIP selling points influence zones determination. A stochastic model coupled with a Geographical Information System was applied for relating resistance and usage density and for detecting city areas of high/low resistance risk. RESULTS: E. coli CIP resistant cluster emergence was detected and significantly related to usage density at a level of 5 to 9 CIP DDDs. There were clustered hot-spots and a significant global spatial variation in the residual resistance risk after allowing for usage density. CONCLUSIONS: There were clustered hot-spots and a significant global spatial variation in the residual resistance risk after allowing for usage density. The usage density of 5-9 CIP DDDs per 1,000 inhabitants within the same influence zone was the resistance triggering level. This level led to E. coli resistance clustering, proving that individual resistance emergence and dissemination was affected by antimicrobial population consumption.

In vitro synergy of ticarcillin/clavulanate in combination with aztreonam and ceftolozane/tazobactam against SPM-1-producing Pseudomonas aeruginosa strains.

Minimal inhibitory concentrations (MICs) of ticarcillin/clavulanic acid (TLc), ceftolozane/tazobactam (C/T), and aztreonam (AT) were determined for 6 SPM-1-producing Pseudomonas aeruginosa (PSA) using Etest® strips and the synergistic effect of such antimicrobials against was evaluated by gradient diffusion strip crossing (GDSC) test. The fraction inhibitory concentration indexes (FICI) were calculated and showed a synergistic (n = 3) and additive (n = 2) effects of TLc + AT against SPM-1 producers, while TLc + C/T combination caused no effect. Average MIC reduction of TLc and AT by GDSC was 3-fold and 2-fold dilutions, respectively. Thus, TLc + AT might be a candidate as a combination therapy to treat SPM-1-producing PSA infections.

Dynamic of High-Risk Acinetobacter baumannii Major Clones in a Brazilian Tertiary Hospital During a Short Time Period.

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant Acinetobacter baumannii strains isolated from a Brazilian tertiary hospital during a 14-day period. The ISAba1-blaOXA-23 structure was found in the chromosome of five isolates, whereas blaOXA-72 was inserted in a 16.6-kb plasmid in two isolates. The presence of ISAba1-blaADC-like justified the high broad-spectrum cephalosporins minimal inhibitory concentrations (MICs) (MIC50, > 512 mg/L) verified in all isolates. Only minocycline (MIC50, ≤ 0.5 µg/mL), polymyxin B (MIC50, 0.5 µg/mL), and tigecycline (MIC50, 0.5 µg/mL) were in vitro active against such isolates. A diversity of other antimicrobial resistance determinants (aph(3')-VIa, aadA1, aac(3')-IIa, strA, strB, sul2, drfA1, mph(E), msr(E), tetB, and floR) was also observed, which may confer resistance to at last six distinct antimicrobial classes. Four distinct pulsed-field gel electrophoresis (PFGE) profiles were observed during the study period, which belonged to ST79/ST258 (n = 2; IC5), ST25/ST229 (n = 2; IC7), ST1 (n = 1; IC1), and ST162/ST235 (n = 1; IC4). Although the ST1 isolate that carried blaOXA-23 and blaOXA-72 was introduced in this hospital setting by a transferred patient, two clonally related ST79/ST258 isolates carrying either one of these carbapenemase encoding genes were recovered from two patients who were hospitalized within the same period of time in the same hospital unit. Finally, a good correlation between PFGE/MLST, blaOXA-51 variant, and single nucleotide polymorphisms was also observed. Here we demonstrated that distinct extensively drug-resistant A. baumannii clones can circulate in the same hospital setting during a short time period, illustrating a very complex epidemiological scenario for this priority pathogen.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Surveillance programs for detection and characterization of emergent pathogens and antimicrobial resistance: results from the Division of Infectious Diseases, UNIFESP.

Several epidemiological changes have occurred in the pattern of nosocomial and community acquired infectious diseases during the past 25 years. Social and demographic changes possibly related to this phenomenon include a rapid population growth, the increase in urban migration and movement across international borders by tourists and immigrants, alterations in the habitats of animals and arthropods that transmit disease, as well as the raise of patients with impaired host defense abilities. Continuous surveillance programs of emergent pathogens and antimicrobial resistance are warranted for detecting in real time new pathogens, as well as to characterize molecular mechanisms of resistance. In order to become more effective, surveillance programs of emergent pathogens should be organized as a multicenter laboratory network connected to the main public and private infection control centers. Microbiological data should be integrated to guide therapy, adapting therapy to local ecology and resistance patterns. This paper presents an overview of data generated by the Division of Infectious Diseases, Federal University of São Paulo, along with its participation in different surveillance programs of nosocomial and community acquired infectious diseases.

Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil.

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.

Evaluation of the susceptibility profiles, genetic similarity and presence of qnr gene in Escherichia coli resistant to ciprofloxacin isolated in Brazilian hospitals.

Increasing quinolone resistance has been reported worldwide, mainly among clinical isolates of Escherichia coli. The objectives of this study were to determine the susceptibility profile, the genetic relatedness, and the prevalence of the qnr gene among ciprofloxacin-resistant Escherichia coli isolated from distinct Brazilian hospitals. A total of 144 ciprofloxacin-resistant Escherichia coli were isolated from 17 Brazilian hospitals between January/2002 and June/2003. The antimicrobial susceptibility testing was performed by microdilution according to NCCLS. The presence of the qnr gene was initially screened by colony blotting, and then confirmed by PCR followed by DNA sequencing. Ninety-five urinary ciprofloxacin-resistant Escherichia coli were further selected for molecular typing by pulsed-field gel electrophoresis (PFGE). Imipenem and meropenem showed the highest susceptibility rates (100.0% for both compounds) followed by amikacin (91.0%) and piperacillin/tazobactan (84.8%). A single ciprofloxacin-resistant Escherichia coli isolate was positive for qnr among the 144 ciprofloxacin-resistant Escherichia coli. Forty-six PFGE patterns were observed among the 95 ciprofloxacin-resistant Escherichia coli type. This study shows that therapeutic options are limited for treatment of ciprofloxacin-resistant Escherichia coli due to the presence of additional mechanisms of antimicrobial resistance, such as ESBL production. The qnr gene was uncommon among ciprofloxacin-resistant Escherichia coli clinical isolates, but its identification might indicate the emergence of this mechanism of quinolone resistance in Brazil. The great genomic variability found among the ciprofloxacin-resistant Escherichia coli highlights the importance of the appropriate use of quinolone to restrict the selection of resistant isolates.

Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System.

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform.

Changing antimicrobial susceptibility patterns among Streptococcus pneumoniae and Haemophilus influenzae from Brazil: Report from the SENTRY Antimicrobial Surveillance Program (1998-2004).

Although antimicrobial resistance rates among Streptococcus pneumoniae and Haemophilus influenzae have increased significantly in most countries in the last years, most studies from Brazil report relatively low resistance rates among these pathogens. In this study, we analyzed the susceptibility patterns of S. pneumoniae and H. influenzae from Brazil during a 7-year period. A total of 829 S. pneumoniae and 718 H. influenzae consecutively collected from 1998 to 2004, mainly from respiratory tract and bloodstream infections, were susceptibility tested by broth microdilution methods against >30 drugs and the results were analyzed by year. Overall, 77.8% of S. pneumoniae strains were considered susceptible (MIC, < or =0.06 microg/ml) to penicillin. Resistance to penicillin (MIC, > or =2 microg/ml) and ceftriaxone (MIC, > or =4 microg/ml) were detected in 7.5 and 0.5% of strains, respectively. The fluoroquinolones, levofloxacin (MIC90) 1 microg/ml) and gatifloxacin (MIC90, 0.5 microg/ml), were active against 99.8% of the isolates tested. Among the other non-beta-lactam drugs tested, the rank order of susceptibility rates was chloramphenicol (98.9%) > clindamycin (96.4%) > erythromycin (90.6%) > tetracycline (69.8%) > trimethoprim/sulfamethoxazole (36.7%). Resistance to penicillin has increased markedly among S. pneumoniae isolates over 7 years (from 2.9 to 11.0%). Additionally, resistance rates against erythromycin, clindamycin, and tetracycline decreased among pneumococcal strains during the same period. S. pneumoniae recovered from pediatric patients (< or =5 years) showed increased penicillin and trimethoprim/sulfametroxazole resistance rates compared to older populations. The rate of ampicillin resistance among H. influenzae was 14.0%, which also corresponds with the beta -lactamase production rate. All H. influenzae isolates were susceptible to amoxicillin/clavulanate (MIC90, 1 microg/ml), ceftriaxone (MIC90, < or =0.008 microg/ml), cefepime (MIC90, 0.12 microg/ml), ciprofloxacin (MIC90, < or = 0.12microg/ml), levofloxacin (MIC90, < or =0.5 microg/ml), and gatifloxacin (MIC90, < or =0.03 microg/ml). Resistance to the antimicrobials tested remained very stable among H. influenzae isolates during the 7-year study period. The continued emerging antimicrobial resistances found in these pathogens (mainly S. pneumoniae) highlight the need for alternative agents for the treatment of infections caused by these species.

Analysis of Intraocular Lens Biofilms and Fluids After Long-Term Uncomplicated Cataract Surgery.

PURPOSE: Postoperative endophthalmitis is a potentially sight-threatening complication of cataract surgery. However, the pathophysiological mechanisms are not completely understood. We sought to study and evaluate the intraocular environment (aqueous and vitreous humors), the capsular tissue, and the intraocular lens (IOL) surfaces of normal eyes after long-term uncomplicated cataract surgery. DESIGN: Experimental laboratory investigation. METHODS: We studied 69 eyes donated for transplantation that had previously undergone cataract surgery with posterior chamber IOL implantation and that had no recorded clinical history of postoperative inflammation. We assessed the intraocular environment (DNA traces and biofilm formation) by microbiological evaluation of intraocular fluids using conventional microbiology and molecular techniques, including assessment for the presence of microbes (biofilm formation) on the IOL surface by scanning electron microscopy and ultrastructural capsular remnants by transmission electron microscopy. RESULTS: Isolated or aggregated cocci were probable in 18.8% of IOL optic surfaces (n = 13) studied by scanning electron microscopy, suggesting the presence of bacterial biofilm. In 3 intraocular fluid samples for IOLs with biofilm, we identified 16S rDNA by polymerase chain reaction and sequencing. No microbial contamination was found in intraocular fluids by conventional microbiological methods. CONCLUSIONS: Our data suggest the possibility of bacterial biofilm formation on the optic surface of IOLs in normal eyes after long-term uncomplicated cataract surgery even in the absence of clinical or subclinical symptoms.