Backyard pigs are a reservoir of zoonotic hepatitis E virus in southern Brazil.

Background: Hepatitis E virus (HEV) is the causative agent of acute hepatitis worldwide. There is no seroprevalence study in backyard farms, which are characterized by suboptimal hygienic conditions in Brazil. We aimed to determine the seroprevalence and genetic diversity of HEV in backyard pigs in Brazil. Methods: Swine serum samples collected in 2012 (n=731) and 2014 (n=713) were analysed. The presence of anti-HEV immunoglobulin G in pig serum was evaluated by indirect enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction was performed and phylogenetic analyses were carried out based on the partial ORF1 and ORF2 coding regions. Results: Anti-HEV antibodies were detected in 77.6% (567/731; 95% confidence interval [CI] 74.5 to 90.6%) of serum samples in 2012 and 65.5% (467/713; 95% CI 62.0 to 69.0%) in 2014. The herd seroprevalence was 91.7% (187/204; 95% CI 91% to 99%) in 2012 and 83.7% (164/196; 95% CI 78% to 89%) in 2014. Further, HEV RNA was detected in 0.8% (6/713) of samples from 2014. Phylogenetic analysis showed three different genotype 3 subtypes with high similarity to human HEV strains. Conclusions: This study showed that backyard pigs are a reservoir of HEV and alerts us to the need to control infection and spillover from backyard farms. GenBank accession numbers: MF438128-MF438135.

Molecular detection and phylogenetic relationship of wild-type strains of canine distemper virus in symptomatic dogs from Uberlândia, Minas Gerais / Detecção molecular e relação filogenética de cepas selvagens do vírus da cinomose canina em cães sintomáticos oriundos de Uberlândia, Minas Gerais

This study investigated the occurrence of canine distemper virus (CDV) by evaluating the presence of viral RNA within urine samples of dogs from Uberlândia, MG, with clinical manifestations suggestive of infection by CDV by targeting the CDV N gene. Of the clinical samples collected ( n =33), CDV viruria was detected in 45.5%. Five dogs died spontaneously; all had characteristic CDV-associated histopathological alterations and demonstrated CDV viruria. Statistical analyses revealed that the age, gender, breed, or the organ system of the dog affected had no influence on the occurrence of canine distemper. Myoclonus and motor incoordination were the most significant neurological manifestations observed. A direct association was observed between keratoconjunctivitis and dogs with CDV viruria. These findings suggest that CDV viruria in symptomatic dogs might not be age related, and that symptomatic dogs can demonstrate clinical manifestations attributed to CDV without viruria identified by RT-PCR. Additionally, the results of the sequence identities analysed have suggested that all Brazilian wild-type strains of CDV currently identified are closely related and probably originated from the same lineage of CDV. Nevertheless, phylogenetic analyses suggest that there are different clusters of wild-type strains of CDV circulating within urban canine populations in Brazil.

A presença do ácido nucleico (RNA) do vírus da cinomose canina (CDV) foi avaliada por meio da amplificação parcial do gene N pela técnica RT-PCR realizada em urina de cães provenientes de Uberlândia, Minas Gerais, que apresentavam sinais clínicos sugestivos de cinomose. Das 33 amostras de urina avaliadas, o CDV foi identificado em 45,5%. Em cinco cães que morreram espontaneamente, além da excreção do CDV na urina, foram observadas alterações histopatológicas associadas à infecção por esse vírus. Análises estatísticas demonstraram que a idade, gênero, raça e o sistema orgânico comprometido dos cães avaliados não exerceram influência no diagnóstico da cinomose canina. Mioclonia e incoordenação motora foram as manifestações neurológicas que apresentaram frequência de ocorrência significativa (P<0,05). Uma associação direta foi observada entre a presença de ceratoconjuntivite e a identificação de virúria pelo CDV. Esses achados sugerem que a excreção do CDV pela urina em cães com sinais clínicos compatíveis com cinomose pode não ser relacionada com a idade do animal, e que animais sintomáticos podem apresentar manifestações clínicas atribuídas ao CDV, porém sem a caracterização de virúria por RT-PCR. Adicionalmente, análises filogenéticas sugerem que várias cepas de CDV podem estar circulando em populações caninas de áreas urbanas no Brasil.

Cerebral cryptococcomas in a cow.

Cerebral cryptococcomas are described in a 5-year-old mixed-breed cow without manifestations of systemic cryptococcosis. Two cryptococcomas were observed grossly. Microscopical examination revealed accumulations of yeast that were morphologically consistent with Cryptococcus neoformans. Immunohistochemistry characterized the organisms as C. neoformans var. grubii.

Effect of cefoxitin and clindamycin on selection of derepressed mutants in Enterobacter cloacae.

The characteristics of an antibiotic that favor its ability to select for resistant bacteria are not completely understood. Otherwise, by the common use of broad-spectrum cephalosporins, resistant strains of several gram-negative species, especially Enterobacter cloacae, have been more frequently isolated. During our studies on beta-lactam resistance in E. cloacae, we observed that the addition of an inhibitor (clindamycin) to a potent inducer (cefoxitin) leads to an enhanced selection of resistant mutants. This could explain the emergence of beta-lactam resistant strains during antibiotic therapy.

Implications of antimicrobial resistance in the empirical treatment of community-acquired respiratory tract infections: the case of macrolides.

Macrolide resistance among pneumococci is increasing worldwide and is associated with increasing macrolide use. Recent studies show that use of macrolides and azalides increases nasopharyngeal carriage of both macrolide-resistant and penicillin-resistant pneumococci. Carriage of a resistant pneumococcus may foster dissemination. The clinical relevance of in vitro resistance has been debated. However, recent data from a matched case-control study showed that 18 (24%) of 76 patients had breakthrough bacteraemia with an erythromycin-resistant pneumococcus while taking a macrolide, whereas none of the 136 matched controls with an erythromycin-susceptible pneumococcal bacteraemia was taking a macrolide (P = 0.0000001). Moreover, five (24%) of 21 patients bacteraemic with the low-level resistant M phenotype and none of the 40 matched controls were taking a macrolide (P = 0.00157). These data indicate that macrolide resistance due to both the efflux and the methylase mechanisms is clinically relevant. Furthermore, they favour guidelines for the empirical treatment of outpatients with community-acquired pneumonia that recommend high-dose oral amoxicillin and reserve coverage of atypical pathogens for selected high-risk populations.

SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains.

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two beta-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 beta-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U. S. isolates had beta-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing beta-lactamases first described in London has now been identified in S. marcescens isolates across the United States.

Evaluation of the Sirscan automated zone reader in a clinical microbiology laboratory.

We compared readings of Kirby-Bauer plates by the Sirscan, an automated image analyzer that measures zone diameters, to those of experienced clinical microbiologists measuring zones with a hand-held caliper interfaced to a computer and with a ruler. To read plates of Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa containing 12 antibiotic disks the Sirscan took 11 s; technologists took 28 s by caliper and 39 s by ruler. Reading times of four different technologists ranged from 22 to 44 s with the caliper and 10 to 12 s with Sirscan. Upon repeated testing zone size variation rarely exceeded 3 mm by caliper and 1 mm by Sirscan. Over a 4-month period, 368 clinical isolates were tested prospectively by both methods in the Clinical Microbiology Laboratory of the Miriam Hospital. There was good correlation of zone sizes for most antibiotics, but Sirscan zone diameter measurements tended to be 3 to 5 mm larger than caliper readings for ciprofloxacin, norfloxacin, aztreonam, erythromycin, clindamycin, and trimethoprim-sulfamethoxazole. Very major errors (resistant by caliper and susceptible by Sirscan) occurred with 10 of 3,770 readings (0.3%), mainly where breakpoint criteria lacked an intermediate zone. They occurred in testing staphylococci with amoxicillin-clavulanate (5 of 127 isolates, 3.9%), pseudomonas with piperacillin (1 of 28, 3.6%), coagulase-negative staphylococci with oxacillin (2 of 74, 2.7%), gram-negative bacilli with cefuroxime (1 of 209, 0.5%), and mixed species with trimethoprim-sulfamethoxazole (1 of 366, 0.3%). The Sirscan zone reader facilitates accurate, fully quantitative susceptibility testing in clinical microbiology laboratories.

Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia.

Nine isolates of Escherichia coli were recovered from seven blood cultures over a period of 3 months from a 19-month-old female with aplastic anemia. Initial isolates were susceptible to extended-spectrum cephalosporins, including ceftazidime (MIC, < or = 0.25 microgram/ml), but gradually became resistant to this drug (MICs, > or = 128 micrograms/ml) and other cephalosporins and the monobactam aztreonam. Molecular typing methods, including plasmid profile analysis, pulsed-field gel electrophoresis, and arbitrarily primed PCR, indicated that the nine isolates were derived from a common ancestor. Dot blot hybridization and PCR analysis of total bacterial DNA using blaSHV- and blaTEM-specific DNA probes and primers identified the presence of a blaTEM beta-lactamase gene in all of the isolates and a blaSHV gene in the isolates with elevated ceftazidime MICs. Isoelectric focusing analysis of crude lysates showed that all nine isolates contained an enzyme with a pI of 5.4 corresponding to the TEM-1 beta-lactamase, and those isolates containing an SHV-type beta-lactamase demonstrated an additional band with a pI of 7.6. The first of the ceftazidime-resistant isolates appeared to hyperproduce the SHV enzyme compared to the other resistant isolates. DNA sequencing revealed a blaSHV-1 gene in the first ceftazidime-resistant isolate and a novel blaSHV gene, blaSHV-8, with an Asp-to-Asn substitution at amino acid position 179 in the remaining four isolates. Three of the ceftazidime-resistant isolates also showed a change in porin profile. The patient had received multiple courses of antimicrobial agents during her illness, including multiple courses of ceftazidime. This collection of blood isolates from the same patient appears to represent the in vivo evolution of resistance under selective pressure of treatment with various cephalosporins.

Comparative susceptibility of clinical isolates producing extended spectrum beta-lactamases to ceftibuten: effect of large inocula.

BACKGROUND: Infections caused by Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) are a growing clinical problem. However, there is wide variation in the level of resistance to third generation beta-lactams conferred by these enzymes. METHODS: We studied 33 Klebsiella pneumoniae and 4 Escherichia coli isolates producing ESBLs obtained from outbreaks in 14 different hospitals and a nursing home in the United States. Microdilution testing with standard (10(4-5) colony-forming units/ml) and large (10(6-7) colony-forming units/ml) inocula, was used to compare the minimum inhibitory concentrations (MICs) of ceftibuten, a novel oral oxyimino beta-lactam, with those of other third generation beta-lactams (cefotaxime, ceftazidime, aztreonam, cefixime, cefpodoxime and cefoxitin). RESULTS: Twenty-seven of the clinical isolates had well-characterized ESBLs of 10 different types, 7 of which produced TEM-1; 1 isolate also produced LXA-1. Two strains produced more than 1 ESBL. The remaining 10 strains produced 8 as yet uncharacterized types of ESBL. With large inocula 73% tested susceptible to ceftibuten, whereas 8 to 22% tested susceptible to the other third generation beta-lactam antibiotics. Ceftibuten MICs increased with higher inocula when tested against strains producing SHV-4 or SHV-5 and, to a lesser extent, strains producing multiple beta-lactamases. Only cefoxitin showed a smaller inoculum effect. CONCLUSION: Ceftibuten merits clinical evaluation in infections caused by bacteria that produce ESBLs.

Net effect of inoculum size on antimicrobial action of ampicillin-sulbactam: studies using an in vitro dynamic model.

To examine the predictable effect of inoculum size on the kinetics of the antimicrobial action of ampicillin-sulbactam, five TEM-1 beta-lactamase-producing Escherichia coli strains were studied in an in vitro dynamic model at two different initial inocula (N0S). All bacteria were exposed to ampicillin-sulbactam in a simulated system reflecting the pharmacokinetic profiles in human tissue after the administration of a single intravenous dose of ampicillin (2 g) plus sulbactam (1 g). Each strain was studied at low (4.0 to 5.2 log CFU/ml) and high (5.0 to 7.1 log CFU/ml) N0S. Despite pronounced differences in susceptibilities, the patterns of the killing curves observed with a given strain at different N0S were similar. As expected, viable bacterial counts increased with inoculum size. Striking visual contrasts in the respective curves for each organism were reflected by the area under the bacterial count-time curve (AUBC) but not by the difference between the N0 and the lowest bacterial counts (Nmin) at the nadir of the killing curve: the N0-associated changes in the AUBC on average were 75%, versus 2.5% for log N0--logNmin. To examine qualitative differences in antimicrobial effects at different N0S (i.e., the net effect of the inoculum), the difference in the high and low N0S was subtracted from each point on the killing curve obtained at the higher N0 for each strain. These adjusted curves were virtually superimposable on the observed killing curves obtained at the lower N0. Moreover, by using adjusted data, the AUBC values were similar at the two inocula, although slight (average, 11%) but systematic increases in the AUBC occurred at high N0S. Thus, there was only a weak net effect of inoculum size on the antibacterial effect of ampicillin-sulbactam. Due to similar slopes of the AUBC-log N0 plots, the antibacterial action at different N0S may be easily predicted by an approximate equation; the predicted AUBCs were unbiased and well correlated with the observed AUBCs (r = 0.997). Compiled data obtained with normalized AUBCs for different strains at different N0S yielded a positive correlation (r = 0.963) between the N0-normalized AUBC and the MIC of ampicillin-sulbactam. The adjustment and normalization procedure described might be a useful tool for revealing the net effect of the inoculum and to predict the inoculum effect if there are no qualitative differences in antimicrobial action at different inocula.

Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics.

beta-Lactamases are the principal mechanism of bacterial resistance to beta-lactam antibiotics. In recent years the number and variety of new beta-lactamases detected has risen at an alarming rate, apparently in response to the clinical use of novel classes of beta-lactam antibiotics. This paper reviews the structure and evolution of beta-lactamases in an attempt to understand the pressures that have contributed to their emergence.

Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae.

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

Predictors of effect of ampicillin-sulbactam against TEM-1 beta-lactamase-producing Escherichia coli in an in vitro dynamic model: enzyme activity versus MIC.

The clinical outcome in patients treated with ampicillin-sulbactam may not always be predictable by disc susceptibility testing or with the MIC as determined with a constant level (4 micrograms/ml) of the beta-lactamase inhibitor (MIC1). The enzyme activities (EA) and the MICs estimated at a constant ratio of ampicillin to sulbactam of 2:1 (MIC2) for 15 TEM-1 beta-lactamase-producing strains of Escherichia coli were examined as alternatives to MIC1 as predictors of the antibacterial effects of this combined drug as studied in an in vitro model which simulates ampicillin-sulbactam pharmacokinetic profiles observed in human peripheral tissues. Integral parameters describing the area under the bacterial count-time curve (AUBC), the area between the normal growth curve, and the killing curve of bacteria exposed to antibiotic (ABBC), and the second parameter expressed as a percentage of its maximal hypothetical value (ABBC/ABBCmax) were calculated. All three parameters correlated well with EA (AUBC, r = 0.93; ABBC, r = -0.88; ABBC/ABBCmax, r = -0.91) and with MIC2 (r = 0.94, -0.94, and -0.95, respectively) but not with MIC1. Both EA and MIC2 can be considered reliable predictors of the antibacterial effect of ampicillin-sulbactam in an in vitro model. These correlations suggest that in vitro kinetic-dynamic models might be useful to reexamine established susceptibility breakpoints obtained with data based on the MIC1 (MICs obtained with constant levels of beta-lactamase inhibitors). These data also suggest that quantitative determinations of bacterial beta-lactamase production and MICs based on the component concentration ratio observed in vivo might be useful predictors of the effect of ampicillin-sulbactam and other beta-lactam-inhibitor combinations.

Controlling vancomycin-resistant enterococci.

After controlling an epidemic of vanB-type vancomycin-resistant Enterococcus faecium (VRE), we contained a subsequent vanA E faecium outbreak by using prospective laboratory-based surveillance, placing patients with VRE in private rooms, requiring the use of both gowns and gloves by all personnel entering the patients' rooms, and conducting prevalence surveys of patients on affected wards.

The growing threat of antibiotic-resistant Streptococcus pneumoniae.

Resistance to penicillin has spread worldwide during the past 25 years. Strains resistant to alternative antibiotics have also emerged. Strains resistant to multiple antibiotics increasingly are isolated worldwide. Recently, isolates of penicillin-resistant S. pneumoniae resistant to cefotaxime and ceftriaxone have caused meningitis. As a result, recommendations for the empiric therapy of pneumococcal infections, especially meningitis, are changing.

IS6770, an enterococcal insertion-like sequence useful for determining the clonal relationship of clinical enterococcal isolates.

Enterococci expressing resistance to antimicrobial agents are increasingly important nosocomial pathogens. Effective strategies to prevent or abort outbreaks of resistant enterococcal infection will rely on an accurate understanding of the mechanisms by which these organisms spread. A 1065-bp insertion-like sequence (IS6770) is present in varying copy numbers in > 90% of enterococcal strains thus far examined. Hybridization patterns resulting from hybridization of enterococcal genomic DNA with an internal IS6770 probe vary considerably between unrelated strains and correlate well with results of pulsed-field gel electrophoresis and field-inversion gel electrophoresis in identifying clonal relationships among enterococcal isolates. IS6770 analysis of several outbreaks of resistant enterococci has confirmed the spread of single resistant clones rather than the emergence of resistance within the resident flora. These results suggest that IS6770 hybridization will be a useful tool for tracing the epidemiology of nosocomial enterococcal infections.