Performed Pulsed-field Gel Electrophoresis Research Articles

Spontaneous Genomic Variation as a Survival Strategy of Nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most important nosocomial human pathogens frequently isolated in bloodstream and medical device-related infections. However, its mechanisms of evolution and adaptation are still poorly explored. To characterize the strategies of genetic and phenotypic diversity in S. haemolyticus, we analyzed an invasive strain for genetic and phenotypic stability after serial passage in vitro in the absence and presence of beta-lactam antibiotics. We performed pulsed-field gel electrophoresis (PFGE) of the culture and analyzed five colonies at seven time points during stability assays for beta-lactam susceptibility, hemolysis, mannitol fermentation, and biofilm production. We compared their whole genomes and performed phylogenetic analysis based on core single-nucleotide polymorphisms (SNPs). We observed a high instability in the PFGE profiles at the different time points in the absence of antibiotic. Analysis of WGS data for individual colonies showed the occurrence of six large-scale genomic deletions within the oriC environ, smaller deletions in non-oriC environ regions, and nonsynonymous mutations in clinically relevant genes. The regions of deletion and point mutations included genes encoding amino acid and metal transporters, resistance to environmental stress and beta-lactams, virulence, mannitol fermentation, metabolic processes, and insertion sequence (IS) elements. Parallel variation was detected in clinically significant phenotypic traits such as mannitol fermentation, hemolysis, and biofilm formation. In the presence of oxacillin, PFGE profiles were overall stable over time and mainly corresponded to a single genomic variant. Our results suggest that S. haemolyticus populations are composed of subpopulations of genetic and phenotypic variants. The maintenance of subpopulations in different physiological states may be a strategy to adapt rapidly to stress situations imposed by the host, particularly in the hospital environment. IMPORTANCE The introduction of medical devices and antibiotics into clinical practice have substantially improved patient quality of life and contributed to extended life expectancy. One of its most cumbersome consequences was the emergence of medical device-associated infections caused by multidrug-resistant and opportunistic bacteria such as Staphylococcus haemolyticus. However, the reason for this bacterium's success is still elusive. We found that in the absence of environmental stresses, S. haemolyticus can spontaneously produce subpopulations of genomic and phenotypic variants with deletions/mutations in clinically relevant genes. However, when exposed to selective pressures, such as the presence of antibiotics, a single genomic variant will be recruited and become dominant. We suggest that the maintenance of these cell subpopulations in different physiological states is an extremely effective strategy to adapt to stresses imposed by the host or the infection environment and might contribute for S. haemolyticus survival and persistence in the hospital.

Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections.

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

152. Molecular Epidemiological Analysis of Relapsed Gram-negative Bloodstream Infection Isolates

Abstract Background Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Many patients experience multiple episodes of GNB-BSI for unclear reasons. This study examines the genetic differences between initial and subsequent isolates of recurrent GNB-BSI. Methods We used a prospective cohort of patients with GNB-BSI at Duke Hospital to identify patients with >1 episode of GNB-BSI due to the same bacterial species. We performed pulsed field gel electrophoresis (PFGE) on paired isolates to determine if the events were Reinfection (paired isolates different) or Relapse (paired isolates genetically identical). We then used whole genome sequencing (WGS) to verify the PFGE findings and explore the genetic similarity between initial and relapsed GNB-BSI isolates. Results Among 1,423 unique patients with GNB-BSI, 60 (4%) experienced recurrent GNB-BSI with the same bacterial species. We performed genotyping (PFGE, followed by WGS) on the paired bacterial isolates from the index and recurrent bloodstream infections for the four most common species in our study population (Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, and Serratia marcescens) (n=48 pairs). We determined that 63% (30/48) of recurrent GNB-BSI episodes were due to relapse and 37% (18/48) were due to reinfection. PFGE correctly identified relapse versus reinfection in 98% (47/48) of cases. The WGS data illustrated the microevolution of relapsed GNB-BSI isolates within patients. When comparing initial to relapsed isolates, we calculated a median of 5 single-nucleotide polymorphisms (SNPs) in relapsed E. coli isolates and 2 SNPs in Klebsiella spp. isolates. Of these SNPs, 37% (E. coli) and 12% (Klebsiella) were non-synonymous changes in coding regions, most commonly genes associated with antimicrobial resistance (AMR), energy metabolism and stress response. Alterations in the AMR profile was common, with 20/30 relapsed isolates demonstrating either loss or acquisition of AMR. Figure 1:Time from initial to recurrent episode of gram-negative bloodstream infection. Relapse (i.e., same bacterial strain noted by green circle) versus reinfection (i.e., different bacterial strain noted by red circle) was determined by pulse field gel electrophoresis followed by WGS. Whole genome sequencing was performed on the initial and recurrent isolate from patients with relapsed GNB-BSI. We identified single nucleotide polymorphisms (SNPs) present in the recurrent isolate, which were not present in the initial isolate. We identified the function of genes containing SNPs in E. coli (n=28 SNPs) and Klebsiella spp. (n=13 SNPs). Synonymous mutations, insertions, deletions, and non-synonymous mutations in hypothetical proteins were excluded. Conclusion Most recurrent GNB-BSI with same species are due to relapse. PFGE has comparable accuracy to WGS to determine reinfection versus relapse. Multiple mutations in genes involved in stress response, AMR and metabolism were identified in relapsed isolates. Disclosures Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds.

678. Outbreak of Shiga Toxin-Producing Escherichia coli Infections at Marine Corps Recruit Depot (MCRD), San Diego and Camp Pendleton, California: October–November, 2017

BackgroundShiga toxin-producing Escherichia coli (STEC) infections are a major cause of foodborne illness and the principal cause of hemolytic-uremic syndrome (HUS). In November 2017, CDC and the US Navy responded to an outbreak of STEC illnesses in military recruits at the Marine Corps Recruit Depot in San Diego (MCRD). We investigated to determine the source of this outbreak and identify prevention and mitigation measures.MethodsIn October 2017, medical staff identified a high number of gastrointestinal (GI) illnesses at MCRD. Recruits with diarrhea submitted stool specimens for culture and/or culture-independent diagnostic testing (CIDT) for GI pathogens. We performed pulsed-field gel electrophoresis (PFGE) on culture isolates. Case-patients were defined as confirmed (PFGE-confirmed STEC infection matching outbreak strains), probable (diagnosis of HUS and/or CIDT evidence of STEC), or suspected (bloody diarrhea). We conducted environmental evaluations of dining facilities, training areas, and barracks. A case–control study was performed using PFGE-confirmed case-patients and platoon-matched controls. We performed product traceback for foods identified as exposure risks by interview or case–control study.ResultsWe identified 64 confirmed, 105 probable, and 91 suspected case-patients. Thirty case-patients required hospitalization and 15 had HUS. Ages ranged from 17 to 28 years (median: 18 years). Poor hygiene practices among recruits and inconsistent cooking temperatures within dining facilities were noted. Forty-three case-patients and 135 controls were interviewed about food, hygiene, and environmental exposures. Consumption of undercooked beef was significantly associated with illness (mOR 2.40, CI 1.04–5.72, P = 0.04). We identified a single ground beef supplier for MCRD, but dining facility records did not document the dates on which specific lots of ground beef were used.ConclusionCase–control analysis and environmental observations suggested undercooked ground beef as a potential source for this outbreak. We recommended the Navy and Marine Corps retain lot information, address food handling concerns, and improve hygiene among recruits.Disclosures All authors: No reported disclosures.

Epidemic characterization and molecular genotyping of Shigella flexneri isolated from calves with diarrhea in Northwest China

BackgroundThe widespread presence of antibiotics resistance genes in pathogenic bacteria can cause enormous problems. Food animals are one of the main reservoirs of intestinal pathogens that pose a potential risk to human. Analyzing the epidemiological characteristics and resistance patterns of Shigella flexneri in calves is necessary for animal and human health.Methods and resultsA total of 54 Shigella flexneri isolates, including six serotypes (1a, 2a, 2b, 4a, 6 and Xv), were collected from 837 fecal samples obtained from 2014 to 2016. We performed pulsed-field gel electrophoresis (PFGE) and applied the restriction enzyme NotI to analyze the genetic relatedness among the 54 isolates and to categorize them into 31 reproducible and unique PFGE patterns. According to the results of antimicrobial susceptibility tests, all 26 Shigella flexneri 2a serotypes were resistant to cephalosporin and/or fluoroquinolones. The genes blaTEM-1, blaOXA-1, and blaCTX-M-14 were detected in 19 cephalosporin-resistant S. flexneri 2a isolates. Among 14 fluoroquinolone-resistant isolates, the aac(6′)-Ib-cr gene was largely present in each strain, followed by qnrS (5). Only one ciprofloxacin-resistant isolate harbored the qepA gene. Sequencing the quinolone resistance determining regions (QRDRs) of the fluoroquinolone-resistant isolates revealed two point mutations in gyrA (S83 L, D87N/Y) and a single point mutation in parC (S80I). Interestingly, two gyrA (D87N/Y) strains were resistant to ciprofloxacin.ConclusionsThe current study enhances our knowledge of Shigella in cattle, although continual surveillance is necessary for the control of shigellosis. The high level of cephalosporin and/or fluoroquinolone resistance in Shigella warns us of a potential risk to human and animal health.

Pulsotype Diversity of Clostridium botulinum Strains Containing Serotypes A and/or B Genes.

Clostridium botulinum strains are prevalent in the environment and produce a potent neurotoxin that causes botulism, a rare but serious paralytic disease. In 2010, a national PulseNet database was established to curate C. botulinum pulsotypes and facilitate epidemiological investigations, particularly for serotypes A and B strains frequently associated with botulism cases in the United States. Between 2010 and 2014 we performed pulsed-field gel electrophoresis (PFGE) using a PulseNet protocol, uploaded the resulting PFGE patterns into a national database, and analyzed data according to PulseNet criteria (UPGMA clustering, Dice coefficient, 1.5% position tolerance, and 1.5% optimization). A retrospective data analysis was undertaken on 349 entries comprised of type A and B strains isolated from foodborne and infant cases to determine epidemiological relevance, resolution of the method, and the diversity of the database. Most studies to date on the pulsotype diversity of C. botulinum have encompassed very small sets of isolates; this study, with over 300 isolates, is more comprehensive than any published to date. Epidemiologically linked isolates had indistinguishable patterns, except in four instances and there were no obvious geographic trends noted. Simpson's Index of Diversity (D) has historically been used to demonstrate species diversity and abundance within a group, and is considered a standard descriptor for PFGE databases. Simpson's Index was calculated for each restriction endonuclease (SmaI, XhoI), the pattern combination SmaI-XhoI, as well as for each toxin serotype. The D values indicate that both enzymes provided better resolution for serotype B isolates than serotype A. XhoI as the secondary enzyme provided little additional discrimination for C. botulinum. SmaI patterns can be used to exclude unrelated isolates during a foodborne outbreak, but pulsotypes should always be considered concurrently with available epidemiological data.

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes

BackgroundMulti-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster.ResultsWe performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains.ConclusionsHere we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

Escherichia coli O104:H4 from 2011 European Outbreak and Strain from Republic of Korea

To the Editor: Beginning in early May 2011, an outbreak caused by Shiga toxin–producing Escherichia coli O104:H4 was reported in Germany and other countries in Europe. In this outbreak, the number of hemolytic uremic syndrome (HUS) cases has been unusually high (1). As of June 9, 2011, a total of 722 cases of HUS, 19 deaths, and 2,745 cases of enterohemorrhagic E. coli (EHEC) infection were reported (2). A case of HUS caused by E. coli O104:H4 was first reported in South Korea in 2004 (3). Because infections caused by E. coli O104:H4 have been reported rarely, interest has arisen in the E. coli O104:H4 strain from South Korea. We characterized the E. coli O104:H4 strain isolated in South Korea (EC0417119) in 2004 and compared it with the E. coli O104:H4 strain associated with the current EHEC outbreak in Europe. The serotype EC0417119, isolated from a patient with HUS in 2004, was reconfirmed as E. coli O104:H4. The strain was positive for stx1 and stx2 by PCR (4) but negative for aggR by PCR (5). In the antimicrobial drug susceptibility test using VITEK 2 AST-N169 test kit (bioMerieux, Marcy L’Etoile, France), the strain was resistant to ampicillin, ampicillin/sulbactam, and trimethoprim/sulfamethoxazole but susceptible to ceftriaxone, cefotaxime, nalidixic acid, and tetracycline. We also performed pulsed-field gel electrophoresis (PFGE) for EC0417119, according to the PulseNet standard protocol (6), and compared its PFGE profile with that of the current outbreak strain E. coli O104:H4, which was obtained from the PulseNet Asia Pacific network. PFGE profiles resolved by either XbaI or BlnI did not match each other. The percentage similarity of XbaI- and BlnI-digested PFGE profiles of the 2 isolates was 75% and 66.7%, respectively, as shown in the Figure. Figure Clustering of A) XbaI- and B) BlnI-digested DNA fragments by pulsed-field gel electrophoresis (PFGE) for Escherichia coli O104:H4 2011 outbreak strain in Europe and isolate obtained in South Korea in 2004. Infections with the EHEC O104 strain were reported several times worldwide. In Europe, such occurrence was rare, and before the current outbreak, the EHEC O104:H4 strain was documented only once in South Korea. For this reason, it was logical to examine the possible relatedness of the EC0417119 strain and the strain causing the current outbreak. However, the EC0417119 strain has many different characteristics compared with the current outbreak strain: not possessing enteroaggregative E. coli determinant, not producing extended-spectrum β-lactamases, and not showing indistinguishable PFGE patterns. In conclusion, there is no evidence that the E. coli O104:H4 strain isolated in South Korea in 2004 is related to the strain that has a caused the massive and unprecedented EHEC outbreak in Europe.

Molecular characterization of toxin A-negative, toxin B-positive variant strains of Clostridium difficile isolated in Korea

A −B + Clostridium difficile strains are prevalent in Korea. We performed pulsed-field gel electrophoresis (PFGE), polymerase chain reaction ribotyping, and toxinotyping in 82 A −B + clinical isolates in Korea. PFGE showed highest discriminatory capability among the 3 methods. By PFGE, persistence of a clone was found, suggesting this clone has adapted to the hospital environment.

Role of Poultry Meat in Sporadic Campylobacter Infections in Bosnia and Herzegovina: Laboratory-based Study

To investigate genetic diversity and specificity of Campylobacter jejuni and Campylobacter coli strains isolated from humans, retail poultry meat, and live farm chickens in Zenica-Doboj Canton, Bosnia and Herzegovina, and identify the role of poultry meat in sporadic Campylobacter infections. We determined the type of Campylobacter species using standard microbiological methods and multiplex polymerase chain reaction (PCR), and performed pulsed field gel-electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) typing of the flaA gene to investigate genetic diversity among the isolates. We isolated C jejuni and C coli from 75 (5.2%) of 1453 samples of consecutive outpatients with sporadic diarrhea; from 51 (34.7%) of 147 samples of poultry meat; and from 15 out of 23 farm chicken samples. The proportion of C coli found among human (30.1%), poultry meat (56.9%), and farm chicken isolates (53.3%), was greater than the proportion of C jejuni. Fourteen and 24 PFGE genotypes were identified among 20 C coli and 37 C jejuni isolates, respectively. Identical PFGE genotypes were found in two cases of human and poultry meat isolates and two cases of poultry meat and farm chicken isolates. Only a minority of human Campylobacter isolates shared identical PFGE type with poultry meat isolates. Although poultry is the source of a certain number of human infections, there may be other more important sources. Further research is required to identify the environmental reservoir of Campylobacter spp responsible for causing human disease and the reason for the high prevalence of C coli human infections in this region.

Insights into the Evolutionary Origin and Genome Architecture of the Unicellular Opisthokonts Capsaspora owczarzaki and Sphaeroforma arctica

Molecular phylogenetic analyses have recently shown that the unicellular amoeboid protist Capsaspora owczarzaki is unlikely to be a nucleariid or an ichthyosporean as previously described, but is more closely related to Metazoa, Choanoflagellata, and Ichthyosporea. However, the specific phylogenetic relationship of Capsaspora to other protist opisthokont lineages was poorly resolved. To test these earlier results we have expanded both the taxonomic sampling and the number of genes from opisthokont unicellular lineages. We have sequenced the protein-coding genes elongation factor 1-alpha (EF1-alpha) and heat shock protein 70 (Hsp70) from C. owczarzaki and the ichthyosporean Sphaeroforma arctica. Our maximum likelihood (ML) and Bayesian analyses of a concatenated alignment of EF1-alpha, Hsp70, and actin protein sequences with a better sampling of opisthokont-related protist lineages indicate that C. owczarzaki is not clearly allied with any of the unicellular opisthokonts, but represents an independent unicellular lineage closely related to animals and choanoflagellates. Moreover, we have found that the ichthyosporean S. arctica possesses an EF-like (EFL) gene copy instead of the canonical EF1-alpha, the first so far described in an ichthyosporean. A maximum likelihood phylogenetic analysis shows that the EF-like gene of S. arctica strongly groups with the EF-like genes from choanoflagellates. Finally, to begin characterizing the Capsaspora genome, we have performed pulsed-field gel electrophoresis (PFGE) analyses, which indicate that its genome has at least 12 chromosomes with a total genome size in the range of 22-25 Mb.

An Outbreak of Salmonella München in Germany Associated with Raw Pork Meat

In summer 2001, an outbreak of Salmonella München occurred in Germany. We conducted descriptive epidemiology and hypothesis-generating interviews among case patients, two retrospective cohort studies, and a case-control study of subout-breaks. We performed pulsed-field gel electrophoresis (PFGE) from selected patient isolates and a limited trace-back investigation for analytical purposes. Four states were consecutively affected: Saxonia (SX), Brandenburg (BB), Berlin (BE), and Baden-Württemberg (BW). Although hypothesis-generating interviews failed to identify a plausible food item, descriptive data and investigations of the suboutbreaks suggested pork meat as a probable source in three states (SX, BB, and BE) but not in BW. The PFGE profiles from isolates of case patients in the first three states were indistinguishable but differed from PFGE profiles of case patients in BW. Trace-back investigation suggested that contamination of pork meat occurred early in the rearing-production chain. This outbreak demonstrates how contamination early in the production process that can yield different end products may complicate multistate outbreaks. Investigation of suboutbreaks and use of the trace-back method as investigational tools may be useful adjuncts in solving the problem of multistate outbreaks.

Susceptibility testing and molecular epidemiology of clinical strains of Bordetella pertussis isolated in Japan from 2001 to 2002

We determined antimicrobial susceptibilities and analyzed molecular epidemiology of 26 strains of Bordetella pertussis clinically isolated and then performed pulsed-field gel electrophoresis (PFGE) in Japan (Japanese Pertussis Surveillance Group Participants), from 2001 to 2002. The MICs of erythromycin, clindamycin, tetracyclines, fluoroquinorones, trimethoprim-sulfamethoxazole and rifampicin of all isolates against these showed 1 microgram/ml or less. Sparfloxacin is the most potent agent, of which the MICs showed 0.008-0.016 microgram/ml. Results of DNA fingerprinting by pulsed-field gel electrophoresis (PFGE) differentiated three types (Type I; 11 strains (42%), type II; 14 strains (54%) and type III; 1 strains (4%)). However, no relation between regions and identical PFGE patterns was found in this study. Further, surveillance of the antimicrobial susceptibilities and molecular epidemiology of B. pertussis will be required.

Outbreak of Salmonella Serotype Anatum Infection Associated With Unpasteurized Orange Juice

In March 1999, a patient was infected with Salmonella serotype Anatum after having consumed unpasteurized orange juice from a manufacturer in Florida. We conducted a cohort study among customers of the manufacturer, performed pulsed-field gel electrophoresis (PFGE) on isolates, and inspected the manufacturing plant. Surveillance data identified three additional patients infected with Salmonella Anatum showing indistinguishable or closely related PFGE patterns. Three of the four patients had consumed orange juice from the same manufacturer. In the cohort study, 6 of 68 persons (9%) who consumed orange juice and/or orange ice cream from the manufacturer were ill, compared with 1 of 47 (2%) who did not. A positive antigen test for Salmonella species and coliform growth in juice samples taken from the production line suggested contamination during the manufacturing process. Commercially produced orange juice should be pasteurized or otherwise processed to achieve equivalent reduction of pathogens.

MOLECULAR TYPING OF ISOLATES FROM AN OUTBREAK ASSOCIATED WITH ERYTHROMYCIN-RESISTANT B. PERTUSSIS. • 1030

Between April 30, and October 1, 1994, 75 probable and confirmed cases of pertussis were identified in Yuma County, AZ. A strain of Bordetella pertussis highly resistant to erythromycin (MIC >64 μg/ml, usual MIC range: 0.02 to 0.1 μg/ml) was isolated from a 2-month-old infant during the outbreak. We performed pulsed-field gel electrophoresis (PFGE) typing on 49 isolates obtained from cases in the affected county and the surrounding area during the same period to (1) determine whether the resistant strain was of a unique genetic type. (2) define the distribution of B. pertussis genetic types in the region, and (3) compare the genetic types of isolates from the region with a random sample of isolates from other states in the U.S.. PFGE and antimicrobial susceptibility testing was performed on 7 isolates from Yuma, 24 from other Arizona counties, and 18 from San Diego County, CA. The 49 isolates yielded 12 distinct PFGE patterns. Two PFGE types. C (N=23) and G (N=11) predominated and comprised 69% of all strains. Five of the 7 isolates from Yuma were PFGE type G compared with 6 of 42 isolates from other counties (OR 15.0, 95% CI=1.9-150). Only the isolate from the index case was resistant to erythromycin and was PFGE type G. For comparison, 87 additional B. pertussis strains isolated during 1993-94 in 7 geographically separate states were typed. Only three of 87 strains isolated elsewhere, compared with 11 of 49 strains from southwestern U.S., were identified as type G (OR 8.1, 95% CI=1.9-39). Four type G strains were isolated during 1989-90 in Missouri; none of the 23 Missouri strains from 1994 were type G. Preliminary data suggest that the mechanism of erythromycin resistance in the index isolate may not involve an easily transmissible genetic element and may be associated with alterations in the chromosomal DNA. These data suggest that B. pertussis can develop resistance to erythromycin. This change in phenotype may not be associated with a detectable change in the PFGE pattern of the strain. Additionally, type G was the dominant strain in the outbreak. This genetic type of B. pertussis has circulated in other parts of the U.S. in the past and thus may not have been imported recently from another country.

Physical and genetic mapping of the telomeric major histocompatibility complex region in man and relevance to the primary hemochromatosis gene ( HFE)

We performed pulsed-field gel electrophoresis (PFGE) on genomic DNA from a radiation hybrid (RH) cell line and constructed a high-resolution physical map of the major histocompatibility complex class I region in 6p21.3, where the gene for primary hemochromatosis (HFE) is believed to be located. Due to the intact microsegment of hemizygous human genomic DNA preserved in the RH cell line, simplified and distinct restriction fragment banding patterns were generated. Using the RH cell line, we were able to extend the physical map of the HLA class I region to about 3000 kb, order the known HLA class I genes from centromere to telomere: HLA-B, -C, -E, (-A, -H, -G), and -F, and orient the HLA-F gene along the chromosome. The proximity of HLA-F to HLA-A was confirmed by linkage and linkage disequilibrium analysis. This study shows that RH cell lines can be useful for constructing long-range physical maps in specific regions of the human genome with PFGE. Physical and genetic mapping studies of this region are consistent with a localization of the HFE gene proximal or distal to HLA-A.