Multiplex polymerase chain reaction for distinguishing Taylorella equigenitalis from Taylorella equigenitalis-like organisms.

It is difficult to distinguish isolates of Taylorella equigenitalis, the cause of contagious equine metritis, from a T. equigenitalis-like organism isolated from asymptomatic donkeys and horses. Although T. equigenitalis is responsible for a severe, contagious disease of the reproductive tract of equids, the T. equigenitalis-like organism, although contagious, does not appear to produce disease. Because of the economic consequences of correctly distinguishing isolates of these 2 microorganisms, a polymerase chain reaction (PCR)-based assay was developed that will distinguish isolates of T. equigenitalis from the T. equigenitalis-like microorganism. The primers used in the PCR assay were designed to amplify unique regions of the gene encoding the 16S ribosomal RNA.

Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus).

Three bacterial isolates that were phenotypically indistinguishable from Taylorella equigenitalis were obtained from the urethral fossae of three male donkeys (Equus asinus), one located in the state of California and the other two in the state of Kentucky, USA. Based on results of pulsed-field gel electrophoresis, the isolate from California differed from the two Kentucky isolates, which were the same. Mares bred artificially (California) or naturally (Kentucky) did not show signs of disease, even though infection with the organism was established in those bred naturally. Mares and, uncharacteristically, all three jacks produced antibodies that reacted in the complement fixation test utilized to identify mares recently infected with T. equigenitalis. Sequence analysis of DNA encoding the 16S rRNA revealed that the gene sequences of these isolates were virtually identical to each other (>99.8% similarity), but different (97.6% similarity) from those of several confirmed isolates of T. equigenitalis. The 16S rDNA sequences of the latter were 100% identical. DNA-DNA hybridization studies revealed a mean hybridization level of 89% between the donkey isolate from California and the donkey isolate from Kentucky. On the other hand, the mean DNA-DNA hybridization level from the donkey isolates with DNA from a strain of T. equigenitalis was 23%. The DNA G+C composition was 37.8 mol% for the two donkey isolates, as well as the strain of T. equigenitalis used in the hybridization studies. These data support our opinion that micro-organisms isolated from the male donkeys are different from T. equigenitalis and it is proposed that they be considered a new species within the genus Taylorella and named Taylorella asinigenitalis sp. nov. The type strain is strain UCD-1T (= ATCC 700933T = LMG 19572T).

Selection bias in epidemiological studies of infectious disease using Escherichia coli and avian cellulitis as an example.

In epidemiological studies of infectious disease, researchers often rely on specific cues of the host, such as clinical signs, as surrogate indicators of pathogen presence. A selection bias would manifest if the specific visual cues used in sampling for the pathogen were not representative of the full range of signs caused by the strains of that pathogen. In our molecular epidemiological studies of Escherichia coli associated with avian cellulitis in broilers, we collect carcasses at the processing plant based on visual cues of lesion morphology. Therefore, the objectives of this study were to: (1) explore the potential impacts of selection bias in an application of infectious disease epidemiology, and (2) utilize a validation protocol to assess the potential for selection bias in our molecular epidemiological studies of E. coli and avian cellulitis. In two different trials, E. coli DNA fingerprints were compared between birds that our observers collected and the birds that the observers missed. Using Fisher's exact tests and simulation models, we determined that the isolates collected by the observers were not significantly different from the isolates missed by the observers (P > 0.60 in both trials). Our method of selecting birds suspected of having cellulitis did not significantly bias our inferences about the population of E. coli associated with cellulitis in the flock. We encourage more investigators to critically assess the relationship of the sample to the target population in epidemiological studies of infectious disease.

Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks.

Avian cellulitis in broiler chickens is primarily caused by Escherichia coli. Previous research found that the E. coli isolates of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. To test the hypothesis that the E. coli associated with cellulitis are endemic in the litter of the broiler house, we designed a study to determine whether E. coli DNA fingerprints associated with cellulitis persist over successive flocks that are grown in the same house. In addition, we assessed the impact of different cleaning and disinfection strategies on this persistence. Two broiler houses were followed on each of five farms over 3-4 flocks. A total of 353 E. coli isolates from cellulitis lesions were analyzed in this study, and 314 of these isolates (89%) were DNA fingerprinted by PFGE. In each ranch, there were several DNA fingerprint patterns that were present over successive flocks, regardless of the cleaning and disinfection strategy utilized. Isolates persisted as long as 191 days, implying that these E. coli are capable of persisting in the broiler house environment for long periods of time. In addition, these E. coli isolates were associated with cellulitis lesions in successive flocks. Thus, the isolates of E. coli that are associated with cellulitis in broiler chickens appear to be endemic in the litter environment of the broiler house.

Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares.

OBJECTIVE: To characterize clinical, serologic, bacteriologic, cytologic, and pathologic endometrial responses of mares to 2 donkey-origin atypical bacterial isolates resembling Taylorella equigenitalis. DESIGN: Prospective in vivo study. ANIMALS: 10 healthy mares. PROCEDURE: Mares in estrus (2/group) were inoculated by intrauterine infusion with 2 isolates of classic T equigenitalis or 2 isolates of atypical Taylorella sp or were sham-inoculated. Bacteriologic, serologic, clinical, uterine, cytologic, and pathologic endometrial responses were assessed 4, 11, 21, 35, and 63 days after inoculation and on day 111 in mares with positive culture results on day 63. RESULTS: One atypical isolate failed to cause infection. The second atypical isolate and both classic T equigenitalis isolates induced similar transient metritis and cervicitis. Both classic isolates and 1 atypical isolate induced anti-T equigenitalis complement-fixing antibodies detectable at day 11. Classic isolates and an atypical isolate provoked intense neutrophilic endometritis followed by a resolving, subacute, neutrophilic-mononuclear endometrial response. The atypical isolate and classic isolates were recovered from the uterus, clitoral fossa, or clitoral sinus of one or both exposed mares for as long as 111 days. CONCLUSIONS AND CLINICAL RELEVANCE: Atypical Taylorella sp infections should be considered as a differential diagnosis of equine infertility in US-origin mares, even those not exposed to stallions from countries where contagious equine metritis occurs. The origins and prevalence of atypical Taylorella sp infection in US horses and donkeys are undetermined.

A statistical model for assessing sample size for bacterial colony selection: a case study of Escherichia coli and avian cellulitis.

A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined.

Bacteria associated with pyothorax of dogs and cats: 98 cases (1989-1998).

OBJECTIVE: To determine the organisms most commonly isolated from pleural fluid from dogs and cats with pyothorax. DESIGN: Retrospective study. ANIMALS: 51 dogs and 47 cats. PROCEDURE: Results of bacteriologic culture of pleural fluid samples obtained by means of thoracentesis were obtained from medical records. To obtain information on in vitro antimicrobial susceptibility of organisms commonly isolated from dogs and cats, records of all dogs and cats examined during 1998 were reviewed, and information was obtained on identity and in vitro antimicrobial susceptibility of aerobic organisms isolated from samples other than urine or urinary tract samples. RESULTS: Median ages of dogs and cats were 4 years. Bacteria were isolated from pleural fluid samples from 47 of 51 (92%) dogs and 45 of 47 (96%) cats. Obligate anaerobic bacteria were isolated from 28 dogs and 40 cats. A mixture of obligate anaerobic and facultative bacteria was isolated from 17 dogs and 20 cats. Samples from cats most often yielded a member of the nonenteric group (most commonly members of the genus Pasteurella), whereas those from dogs more often yielded a member of the family Enterobacteriaceae (most commonly E coli). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that antimicrobial agents chosen for the initial treatment of dogs and cats with pyothorax should be active against a mixture of obligate anaerobic and facultative bacteria.

Spatial heterogeneity of Escherichia coli DNA fingerprints isolated from cellulitis lesions in chickens.

Avian cellulitis in broiler chickens is characterized by subcutaneous lesions that result in economic losses because of the partial or complete condemnation of the carcasses at processing. Escherichia coli is the primary causative agent of this condition. Previous research with a biotyping system found that the E. coli of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. The objective of our study was to analyze the genetic variability of E. coli isolates associated with cellulitis. We analyzed the genetic relatedness of the isolates in relation to the houses, ranches, and complexes in which the broilers were grown. This analysis enabled us to assess the spatial heterogeneity, or genetic diversity on a spatial scale, of the isolates. Forty-nine broilers with cellulitis lesions were necropsied. These broilers came from six houses on four ranches on three complexes that had been placed with chicks from the same hatchery within a 2-wk period. Isolates of E. coli from the lesions were DNA fingerprinted by pulsed-field gel electrophoresis. Relatedness among isolates was determined with the Dice coefficient and an unweighted pair group method with average linkages cluster analysis. The complexes possessed isolates with a variety of DNA fingerprints, yet each complex appeared to have isolates with a unique set of DNA fingerprints. Isolates from the same complex tended to form clusters with similarity coefficients greater than 90%. Isolates from different complexes were genetically distinct. This heterogeneity at the level of the complex suggests that isolates were not disseminated from a source common to the complexes. The spatial heterogeneity of the E. coli isolates in this study implies an endemic population of cellulitis-associated E. coli exists in the broiler house environment.

Antimicrobial susceptibility of Nocardia nova isolated from five cats with nocardiosis.

From 1992 through 1997, 5 cats were admitted to the hospital because of chronic, nonhealing lesions containing draining tracts. Exudate from 2 of the 5 cats contained macroscopically visible granules. On the basis of cytologic findings, lesions were described as pyogranulomatous. Degenerative neutrophils and activated macrophages, along with slender, branching, gram-positive, partially acid-fast microorganisms, were observed in stained smears of exudates obtained from all 5 affected cats. Nocardia nova was found in pure culture from all affected sites. Most isolates were susceptible to ampicillin, aminoglycosides (ie, amikacin, kanamycin), tetracyclines (ie, doxycycline, minocycline), macrolides (ie, erythromycin, clarithromycin), imipenem, sulfisoxazole, and trimethoprim-sulfamethoxazole. Other antimicrobials were less effective, and these included amoxicillin-clavulanic acid, the cephalosporins (ie, cefotaxime, ceftizoxime, ceftriaxone), and some aminoglycosides (ie, gentamicin, tobramycin). Four of the 5 cats were successfully treated, 3 with a trimethoprim-sulfonamide combination, and 1 with clarithromycin. The outcome of treatment of the fifth cat is unknown. Findings in this report may be useful in diagnosis and treatment of nocardiosis caused by N nova in cats.

Wound contamination and antimicrobial susceptibility of bacteria cultured during total ear canal ablation and lateral bulla osteotomy in dogs.

OBJECTIVE: To detect contamination of wound sites from surgical handling of excised tissues during total ear canal ablation and lateral bulla osteotomy in dogs, and to compare susceptibility of bacterial isolates to cefazolin with susceptibility to other antimicrobial agents. DESIGN: Prospective clinical study. ANIMALS: 13 dogs. PROCEDURE: Dogs were treated surgically for otitis externa and media via total ear canal ablation and lateral bulla osteotomy. Specimens for aerobic bacterial culture were obtained from SC tissue immediately following skin incision, tissues excised from the osseous bulla (after transection of the horizontal ear canal and lateral bulla osteotomy), and from SC tissue prior to skin closure. Antimicrobial susceptibility of bacterial isolates to various antibiotics was determined by use of a broth dilution assay. RESULTS: There was a significant association between isolation of Streptococcus canis and Escherichia coli from specimens from the osseous bulla and specimens from the SC tissues prior to skin closure, indicating contamination of the SC tissues during surgery. Seventy percent of bacterial isolates were susceptible to cefazolin. CLINICAL IMPLICATIONS: Measures to limit bacterial contamination resulting from tissue handling during total ear canal ablation and lateral bulla osteotomy are necessary. Bacteriologic culture of tissue of the osseous bulla and determination of antimicrobial susceptibility are recommended. Administration of cefazolin alone may not be efficacious for antimicrobial prophylaxis.

Evaluation of methods to diagnose Clostridium perfringens-associated diarrhea in dogs.

OBJECTIVE: To assess the prevalence of Clostridium perfringens enterotoxin in feces of dogs with and without diarrhea, and to compare the use of microbial cultures from fecal specimens and evaluation of stained fecal smears for endospores with the presence of enterotoxin as tools for diagnosing C perfringens-associated diarrhea. DESIGN: Prospective study. ANIMALS: 144 dogs representing hospitalized dogs with (n = 41) or without (50) diarrhea, and clinically normal dogs treated as outpatients (53). PROCEDURE: Fresh fecal specimens from all dogs were examined as Gram-stained fecal smears to determine numbers of Gram-positive spore-forming rods/100x objective field. Enterotoxin was assayed directly by use of a reverse passive latex agglutination assay. Fecal specimens were plated directly to prereduced egg yolk agar plates and incubated overnight at 37 C in an anaerobic chamber. At 24 hours, up to 3 lecithinase-positive colonies were subcultured to Brucella blood agar to evaluate for double zone hemolysis. Colonies with double zone hemolysis were tested for aerotolerance and Gram-stained. RESULTS: A significant difference was not detected among groups with respect to the presence of C perfringens as determined by culture, the presence of endospores, and the reaction patterns of fecal enterotoxin assays. An association was not found between number of endospores and the presence of fecal enterotoxin. CLINICAL IMPLICATIONS: The presence of C perfringens enterotoxin in feces of dogs, as detected by the latex agglutination assay used in this study, correlates poorly with the number of fecal endospores, regardless of the dog's clinical status.

Outbreak of fatal salmonellosis in cats following use of a high-titer modified-live panleukopenia virus vaccine.

A 14-week-old kitten from a private cattery was examined because of an acute onset of recumbency and epistaxis 10 days after receiving a high-titer modified-live virus vaccine containing panleukopenia virus, calicivirus, and herpesvirus components. The kitten died the following day, and intestinal crypt necrosis; hepatic, splenic, and lymph node inflammation and necrosis; and pneumonia were seen at necropsy. Salmonella typhimurium was isolated from mesenteric lymph nodes and the spleen. The breeder reported that 4 other kittens had died in the previous month, each within 1 to 2 weeks after being vaccinated with the same modified-live virus vaccine. Carcasses of 3 kittens were available for examination, and Salmonella sp was isolated from mesenteric lymph nodes of all 3. Villus crypt necrosis and secondary fibrosis were also found. Three of the remaining 12 kittens in the cattery were also found to be shedding Salmonella sp in their feces. Clinical and pathologic findings in these kittens were likely attributable to salmonellosis and panleukopenia, and suggest that mild immunosuppression induced by vaccination could have facilitated development of fatal salmonellosis in subclinical carrier kittens. However, we cannot prove that vaccination actually played any role.

Comparative molecular characterization of Corynebacterium pseudotuberculosis of different origin.

Ribotyping and susceptibility to 17 antimicrobial agents were used to compare 37 isolates of Corynebacterium pseudotuberculosis (28 from horses, 1 from cattle, 3 from sheep and 5 from goats) derived from various types of lesions, and different geographic locations. According to the presence of nitrate reductase, all but one isolate from horses reduced nitrate (nitrate-positive), whereas all isolates from sheep and goats were unable to reduce nitrate (nitrate-negative). The ribotype of the nitrate-negative isolate from a horse with ulcerative lymphangitis was identical to all the other isolates from horses, and different than the ribotype of nitrate-negative isolates from sheep and goats. Ribotyping with one of the restriction endonucleases, Apa 1, revealed differences between, but not within, the two biotypes. However, ribotyping with Pst 1 endonuclease revealed one variant within the equine biotype and one variant within the ovine biotype. The minimum inhibitory concentration (MIC; microgram/ml) of antimicrobial agents against isolates from nitrate-negative and nitrate-positive groups was very similar, with the exception of isolates from sheep and goats which had a higher MIC for amikacin than isolates from horses and cattle.

Assessment of spatial and temporal clustering of ampicillin- and tetracycline-resistant strains of Pasteurella multocida and P haemolytica isolated from cattle in California.

OBJECTIVE: To determine whether ampicillin- and tetracycline-resistant strains of Pasteurella multocida and P haemolytica isolated from California cattle with pneumonia were spatially and temporally clustered and to compare overall estimates of percentages of these isolates resistant to these antimicrobials with estimates obtained on the basis of regional and temporal information. DESIGN: Epidemiologic study. SAMPLE POPULATION: Records of P multocida and P haemolytica isolates obtained from lung or tracheal wash samples collected from California cattle with pneumonia between July 1, 1991 and July 31, 1996. Only isolates obtained from samples submitted by dairies and calf ranches were used. PROCEDURE: Spatial clustering of ampicillin- and tetracycline-resistant isolates was assessed by use of nearest-neighbor and Cuzick and Edwards' analyses. Linear clustering along a north-south line was assessed by use of runs and maximum length of runs tests. Temporal clustering was assessed by use of scan tests. Spatial-temporal clustering was assessed by use of Barton's method. Regional estimates of percentages of P multocida and P haemolytica resistant to ampicillin or tetracycline were calculated. RESULTS: There was significant spatial clustering of resistant isolates and significant linear clustering along a north-south line. Significant differences in regional estimates of percentages of antimicrobial-resistant isolates were found. CLINICAL IMPLICATIONS: Results support the hypothesis that antimicrobial-resistant organisms can be clustered at the local level and reinforce the need to establish regional estimates of percentages of bacterial isolates that will be susceptible to commonly used antimicrobials.

Detection of Pasteurella multocida-specific DNA in turkey flocks by use of the polymerase chain reaction.

A polymerase chain reaction (PCR)-based assay using primers constructed to amplify the gene (psl) encoding the P6-like protein (Psl) of Pasteurella multocida was developed. After Southern blotting and hybridization with psl, the assay (PCR-H) was found to be specific (it did not detect a variety of other avian bacterial pathogens) and sensitive (detected > or = 10 P. multocida organisms or > or = 24 femtograms of extracted P. multocida DNA). Samples were collected from the oropharynx of randomly selected birds housed on premises that had recently experienced an outbreak of avian cholera (outbreak farms) or from birds housed on premises that had not reported an outbreak of this disease during the preceding 12 mo (control farms). The PCR-H assay detected 11 infected turkeys out of a total of 178 sampled on six outbreak farms as compared with isolation of P. multocida from 23 turkeys by using mouse inoculation. Neither method detected P. multocida in samples collected from 174 turkeys sampled on six control farms. Statistical analysis using the Kappa test demonstrated that the results of the two tests showed poor agreement from five outbreak flocks (K = 0, 0, 0, 0.35, 0.47) and strong agreement from one outbreak flock (K = 0.89). Combined results from the outbreak flocks showed poor agreement (K = 0.49) between the two methods.

Organisms isolated from dogs and cats with anaerobic infections and susceptibility to selected antimicrobial agents.

OBJECTIVE: To determine the prevalence of obligate anaerobic bacteria in bacterial infections in dogs and cats and susceptibility to selected antimicrobial agents. DESIGN: Case series. SAMPLE POPULATION: Specimens from 1,267 dogs and 243 cats. PROCEDURE: Standard anaerobic and aerobic bacterial culture methods were used. Anaerobic isolates were tested for susceptibility to selected antimicrobial agents. RESULTS: Obligate anaerobic bacteria were isolated from 199 (15.7%) and 69 (28.4%) specimens obtained from dogs and cats, respectively. More than half of the specimens that contained obligate anaerobic bacteria were from draining tracts (exclusively dogs), pleural fluid, abscesses, bones, the respiratory tract, or the abdominal cavity. The most commonly isolated obligate anaerobic bacteria (approx 70% of all isolates) were Bacteroides spp, Peptostreptococcus spp, Fusobacterium spp, and Porphyromonas spp. Eighty percent of the specimens that contained obligate anaerobic bacteria also contained facultative anaerobic or aerobic organisms. The organisms most commonly isolated in association with obligate anaerobic bacteria were members of the family Enterobacteriaceae (Escherichia coli was the most common), Pasteurella spp, and Staphylococcus intermedius. Ninety-seven obligate anaerobic isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, chloramphenicol, clindamycin, and metronidazole. All were susceptible to amoxicillin-clavulanic acid and chloramphenicol, and most were susceptible to metronidazole. Only 71% of the Bacteroides isolates were susceptible to ampicillin, and only 83% were susceptible to clindamycin. Only 80% of the Clostridium isolates were susceptible to clindamycin, but all were susceptible to ampicillin. CLINICAL IMPLICATIONS: Data on sites and conditions from which anaerobic bacteria are commonly isolated, along with results of susceptibility testing, may be useful in designing antimicrobial treatment regimens.

Pasteurella multocida enters polarized epithelial cells by interacting with host F-actin.

We investigated the interaction of an avian strain of Pasteurella multocida with the cytoskeleton of MDCK cells, which formed a polarized epithelium when grown on type I collagen coated filters. Bacteria were incubated with MDCK cells for 30 min. 2, 4 and 6 hours and their location and association with the cell cytoskeleton determined by double-label immunofluorescence confocal microscopy. Cells were stained with a polyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) actin. Confocal microscopy revealed that bacteria entered the cells by 30 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Electron microscopy demonstrated that by 2 hours, each bacterium was associated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection disrupted the actin cytoskeleton and inhibited entry of P. multocida.

Microbiological study of transtracheal aspirates from dogs with suspected lower respiratory tract disease: 264 cases (1989-1995).

OBJECTIVE: To determine the most commonly isolated bacterial species associated with lower respiratory tract disease of dogs and to determine susceptibility of these isolates to antimicrobial agents. DESIGN: Retrospective case series. SAMPLE POPULATION: Transtracheal aspirates from 264 dogs with clinical evidence of lower respiratory tract disease. PROCEDURE: Records of microbiological analyses of transtracheal aspirates obtained from dogs with clinical evidence of lower respiratory tract disease were reviewed. Analyses performed included bacterial culture (anaerobic and aerobic organisms) and susceptibility testing (aerobic organisms). The medical record of each affected dog was evaluated to determine signalment and underlying condition. RESULTS: Bacteria were isolated from 116 of 264 (44%) samples, and 203 bacterial species were identified. Most (57%) of the samples from which bacteria could be isolated contained a single species, whereas 43% yielded cultures of mixed species. Bacterial species belonging to the family Enterobacteriaceae (particularly Escherichia coli) were isolated most commonly (45.7% of samples contained members of this group), followed by members of the genus Pasteurella (22.4%), obligate anaerobes (21.6%), beta-hemolytic Streptococcus (12.1%), Bordetella bronchiseptica (12.1%), nonhemolytic Streptococcus/Enterococcus sp group (12.1%), coagulase-positive Staphylococcus (9.5%), and Pseudomonas sp (7.8%). The most active antimicrobial drugs (inhibiting > 90% of the isolates) for aerobic microorganisms encountered most often (E. coli and Pasteurella sp) included amikacin, ceftizoxime sodium, enrofloxacin, and gentamicin sulfate. CLINICAL IMPLICATIONS: Amikacin, ceftizoxime, enrofloxacin, and gentamicin may be rational choices for treatment of suspected infectious lower respiratory tract disease of dogs, before identification of the causative agent(s) and before results of susceptibility tests become available.