Genomic Epidemiology of Salmonella enterica Circulating in Surface Waters Used in Agriculture and Aquaculture in Central Mexico.

Salmonella enterica can survive in surface waters (SuWa), and the role of nonhost environments in its transmission has acquired increasing relevance. In this study, we conducted comparative genomic analyses of 172 S. enterica isolates collected from SuWa across 3 months in six states of central Mexico during 2019. S. enterica transmission dynamics were assessed using 87 experimental and 112 public isolates from Mexico collected during 2002 through 2019. We also studied genetic relatedness between SuWa isolates and human clinical strains collected in North America during 2005 through 2020. Among experimental isolates, we identified 41 S. enterica serovars and 56 multilocus sequence types (STs). Predominant serovars were Senftenberg (n = 13), Meleagridis, Agona, and Newport (n = 12 each), Give (n = 10), Anatum (n = 8), Adelaide (n = 7), and Infantis, Mbandaka, Ohio, and Typhimurium (n = 6 each). We observed a high genetic diversity in the sample under study, as well as clonal dissemination of strains across distant regions. Some of these strains are epidemiologically important (ST14, ST45, ST118, ST132, ST198, and ST213) and were genotypically close to those involved in clinical cases in North America. Transmission network analysis suggests that SuWa are a relevant source of S. enterica (0.7 source/hub ratio) and contribute to its dissemination as isolates from varied sources and clinical cases have SuWa isolates as common ancestors. Overall, the study shows that SuWa act as reservoirs of various S. enterica serovars of public health significance. Further research is needed to better understand the mechanisms involved in SuWa contamination by S. enterica, as well as to develop interventions to contain its dissemination in food production settings. IMPORTANCE Surface waters are heavily used in food production worldwide. Several human pathogens can survive in these waters for long periods and disseminate to food production environments, contaminating our food supply. One of these pathogens is Salmonella enterica, a leading cause of foodborne infections, hospitalizations, and deaths in many countries. This research demonstrates the role of surface waters as a vehicle for the transmission of Salmonella along food production chains. It also shows that some strains circulating in surface waters are very similar to those implicated in human infections and harbor genes that confer resistance to multiple antibiotics, posing a risk to public health. This study contributes to expand our current knowledge on the ecology and epidemiology of Salmonella in surface waters.

Evaluation of a Bead-Based Salmonella Molecular Serotyping Method for Salmonella Isolated from Food and Environmental Samples.

Salmonella is a leading cause of foodborne illness worldwide, and foods containing Salmonella (except raw meat and poultry products) are considered adulterated. Serotyping of Salmonella is an essential part of surveillance and investigation of outbreaks. This study evaluated a bead-based Salmonella molecular serotyping (SMS) method, which included the O-group 1, H-antigen, alternate target, and O-group 2 assays, compared with traditional serotyping. Salmonella was isolated from food, pet food, and environmental samples or were reference strains. A total of 572 isolates were analyzed by using two formats of the SMS method in comparison with traditional methods: 485 were analyzed by using Radix SMS (a custom user-mixed format), 218 were analyzed by using Luminex SMS (a commercial kit format), and 131 of the total isolates were analyzed by both formats for comparison. The SMS method was evaluated on the basis of the successful identification of antigens by the probes included in the method. The method identified 550 (96.2%) isolates as expected, 6 (1.0%) isolates were not identified as initially expected but were shown to be correctly identified by SMS after reanalysis by traditional serotyping, and 16 (2.8%) isolates not identified as expected possessed an antigen that should have been detected by the method but was not. Among the isolates considered correctly identified, 255 (44.6%) were identified to a single serovar, 44 (7.7%) required additional biochemical testing to differentiate variants or subspecies, and 251 (43.9%) were partially serotyped because probes for some antigens were not in the assay or had allelic variation for known serovars. Whole genome sequencing, SeqSero, and the Salmonella In Silico Typing Resource gave added confirmation for three isolates. Addition of the O-group 2 assay enabled the identification of 55 (9.6%) of 572 isolates. The SMS method could fully or partially serotype most isolates within a day. The SMS method should be a valuable tool when faster screening methods are needed, such as outbreaks and screening large numbers of environmental isolates.

Insect exclusion limits variation in bacterial microbiomes of tomato flowers and fruit.

AIMS: The effect of insect exclusion via netting on bacterial microbiota associated with field-grown tomato fruit and flowers was evaluated. METHODS AND RESULTS: Amplicon-based bacterial community profiling from insect-exposed plants and plants wrapped in nylon mosquito netting was conducted on total DNA extracted from tomato flower and mature unripe fruit washes. The V1-V3 region of the 16S rRNA gene was sequenced using Illumina MiSeq and analysed using qiime ver. 1.8. The carposphere supported significantly more phylogenetic diversity (PD) compared to the anthosphere, as measured by operational taxonomic unit richness (P = 0·001) and Faith's PD (P = 0·004). Flowers and fruit hosted distinct bacterial community structures (R2 = 0·27, P = 0·001), with specific taxonomic differences in taxa that included the Xanthomonadaceae (higher in flowers), and the Pseudomonadaceae, Methylobacteriaceae and Rhizobiales (higher in fruit) (FDR-P < 0·05). Bacterial community profiles of netted plants were overall statistically similar to non-netted plants for both flowers and fruit (P > 0·10). However, less variation between samples was observed among flowers (~50% less, P = 0·004) and green fruit (~10% less, P = 0·038) collected from netted than non-netted plants. CONCLUSION: Insects may introduce or augment variability in bacterial diversity associated with tomato flowers and potentially green fruit surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to knowledge on microbiome dynamics of the tomato holobiont. Deciphering drivers of bacterial diversity and community structure of fruit crops could reveal processes important to agricultural management, such as competitive exclusion of pathogens and priming of plant defense mechanisms.

Evaluation of 3M Molecular Detection System and ANSR Pathogen Detection System for rapid detection of Salmonella from egg products.

Isothermal amplification assay is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of the 3M Molecular Detection System (MDS) and ANSR Pathogen Detection System (PDS) for the detection of Salmonella in egg products as compared to the Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method and a modified culture method (3M MDS and ANSR PDS preferred method). Two Salmonella ser. Enteritidis (18579, PT4; CDC_2010K_1441, PT8), one Salmonella ser. Heidelberg (607310-1), and one Salmonella ser. Typhimurium (0723) isolates were used in this study. Seven wet egg products and 13 dry egg products were inoculated with these strains individually at 1 to 5 CFU/25 g. One set of test portions was prepared following FDA BAM procedures [with lactose broth (LB) as pre-enrichment broth]. Another set of test portions was prepared using buffered peptone water (BPW) as pre-enrichment broth, as instructed by the 2 detection systems. Results from 3M MDS and ANSR PDS were 100% in agreement with their BPW-based culture method results. When LB was used as pre-enrichment broth, the number of Salmonella positive test portions (80 tested), identified with the BAM, 3M MDS, and ANSR PDS, were 63, 61, and 60, respectively. In conclusion, both 3M MDS and ANSR PDS Salmonella assays were as effective as their BPW based culture methods and were equivalent to the BAM culture method for the detection of Salmonella in egg products. These sensitive isothermal assays can be used as rapid detection tools for Salmonella in egg products provided that BPW is used as pre-enrichment broth.

Simultaneous Presence of Insertion Sequence Excision Enhancer and Insertion Sequence IS629 Correlates with Increased Diversity and Virulence in Shiga Toxin-Producing Escherichia coli.

Although new serotypes of enterohemorrhagic Escherichia coli (EHEC) emerge constantly, the mechanisms by which these new pathogens arise and the reasons emerging serotypes tend to carry more virulence genes than other E. coli are not understood. An insertion sequence (IS) excision enhancer (IEE) was discovered in EHEC O157:H7 that promoted the excision of IS3 family members and generating various genomic deletions. One IS3 family member, IS629, actively transposes and proliferates in EHEC O157:H7 and enterotoxigenic E. coli (ETEC) O139 and O149. The simultaneous presence of the IEE and IS629 (and other IS3 family members) may be part of a system promoting not only adaptation and genome diversification in E. coli O157:H7 but also contributing to the development of pathogenicity among predominant serotypes. Prevalence comparisons of these elements in 461 strains, representing 72 different serotypes and 5 preassigned seropathotypes (SPT) A to E, showed that the presence of these two elements simultaneously was serotype specific and associated with highly pathogenic serotypes (O157 and top non-O157 Shiga toxin-producing Escherichia coli [STEC]) implicated in outbreaks and sporadic cases of human illness (SPT A and B). Serotypes lacking one or both elements were less likely to have been isolated from clinical cases. Our comparisons of IEE sequences showed sequence variations that could be divided into at least three clusters. Interestingly, the IEE sequences from O157 and the top 10 non-O157 STEC serotypes fell into clusters I and II, while less commonly isolated serotypes O5 and O174 fell into cluster III. These results suggest that IS629 and IEE elements may be acting synergistically to promote genome plasticity and genetic diversity among STEC strains, enhancing their abilities to adapt to hostile environments and rapidly take up virulence factors.

Comparison of different preenrichment broths, egg:preenrichment broth ratios, and surface disinfection for the detection of Salmonella enterica ssp. enterica serovar Enteritidis in shell eggs.

Salmonella enterica ssp. enterica serovar Enteritidis is the leading reported cause of Salmonella infections. Most Salmonella Enteritidis infections are associated with whole shell eggs and egg products. This project attempted to lay the foundation for improving the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella Enteritidis in shell eggs. Two Salmonella Enteritidis isolates were used for comparisons among different preenrichment and enrichment media and for the evaluation of egg:preenrichment broth ratios for the detection of Salmonella Enteritidis in shell eggs. The effect of surface disinfection on the detection of Salmonella Enteritidis in shell eggs was also investigated. The results indicated that tryptic soy broth (TSB) was similar to TSB plus ferrous sulfate, but significantly (α = 0.05) better than nutrient broth, Universal Preenrichment broth, and buffered peptone water when used for preenrichment of Salmonella in shell eggs. Salmonella Enteritidis populations after enrichment with Rappaport-Vassiliadis broth were 0.40 to 1.11 log cfu/mL of culture lower than those in preenrichment cultures. The reduction was statistically significant (α = 0.05). Egg:broth ratios at 1:9 and 1:2 produced significantly (α = 0.05) higher Salmonella Enteritidis populations after preenrichment with TSB with inoculum levels at 4 cfu/100 g of eggs and 40 cfu/1,000 g of eggs than the ratio at 1:1. Salmonella Enteritidis populations in TSB preenrichment cultures of shell eggs surface-disinfected with 70% alcohol:iodine/potassium iodide solution and untreated control were 9.11 ± 0.11 and 9.18 ± 0.05 log cfu/mL, respectively, for SE 13-2, and 9.20 ± 0.04 and 9.16 ± 0.05 log cfu/mL, respectively, for SE CDC_2010K_1543. Surface disinfection of eggs did not reduce the sensitivity of detection of Salmonella Enteritidis in liquid eggs. These results could improve the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella in shell eggs by simplifying the preenrichment medium and changing the sample handling before enrichment.

Eight years of Legionnaires' disease transmission in travellers to a condominium complex in Las Vegas, Nevada.

Travel is a risk factor for Legionnaires' disease. In 2008, two cases were reported in condominium guests where we investigated a 2001 outbreak. We reinvestigated to identify additional cases and determine whether ongoing transmission resulted from persistent colonization of potable water. Exposures were assessed by matched case-control analyses (2001) and case-series interviews (2008). We sampled potable water and other water sources. Isolates were compared using sequence-based typing. From 2001 to 2008, 35 cases were identified. Confirmed cases reported after the cluster in 2001-2002 were initially considered sporadic, but retrospective case-finding identified five additional cases. Cases were more likely than controls to stay in tower 2 of the condominium [matched odds ratio (mOR) 6·1, 95% confidence interval (CI) 1·6-22·9]; transmission was associated with showering duration (mOR 23·0, 95% CI 1·4-384). We characterized a clinical isolate as sequence type 35 (ST35) and detected ST35 in samples of tower 2's potable water in 2001, 2002, and 2008. This prolonged outbreak illustrates the importance of striving for permanent Legionella eradication from potable water.

Genetic and phylogenetic evidence for misidentification of Vibrio species within the Harveyi clade.

AIM: This report describes the use of a six-gene multi-locus sequence analysis (MLSA) to correctly identify Vibrio strains of the Harveyi clade. METHODS AND RESULTS: Vibrio isolates were characterized using a six housekeeping gene MLSA. The study provided evidence supporting: (i) a substantial number of reference strains maintained within commercial culture collections are misidentified taxonomically at the species level; (ii) two V. alginolyticus subclades retain species-level divergence; and (iii) V. communis and V. owensii likely are the same species. CONCLUSION: A significant number (n = 10) of Harveyi clade Vibrio strains have been inaccurately identified, including evidence that V. communis and V. owensii strains, two recently discovered species assigned to the Harveyi clade, comprise a single species. SIGNIFICANCE AND IMPACT OF THE STUDY: As Harveyi clade vibrios have an enormous impact on human and aquatic animal health, it is of paramount importance to identify members of the Harveyi clade correctly.

Genome sequence of Cronobacter sakazakii E899, a strain associated with human illness.

Cronobacter has caused numerous illnesses in neonates, infants, and children. Here we report the draft genome of Cronobacter sakazakii E899. Whole-genome sequence analysis of Cronobacter strains provides a tool for understanding the genomic regions specific to each individual species.

Genome sequences of Listeria monocytogenes strains J1816 and J1-220, associated with human outbreaks.

Listeria monocytogenes has caused numerous human outbreaks. Here we report draft genomes of L. monocytogenes J1816 and J1-220, which belong to epidemic clones II and IV, respectively. Whole-genome sequence analysis of these strains provides a tool for studying the short-term evolution of these epidemic clones.

Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and ß-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).

The reaction between plasmin and C1-inhibitor results in plasmin inhibition by the serpin mechanism.

C1-inhibitor is an important inhibitor of plasma kallikrein and C1, but also has inhibitory activity against numerous other plasma proteinases such as plasmin. The relevance of plasmin inhibition by the C1-inhibitor has been debated, with some evidence showing that plasmin causes significant proteolysis of C1-inhibitor. In the present study, we show that C1-inhibitor in its native state will inhibit plasmin without being significantly degraded, in a manner typical of all serpin reactions. However, if C1-inhibitor is in a denatured polymeric state (as can easily occur during storage, or as produced by heating of the native protein), it will be extensively degraded by plasmin. In addition, we show that hydrophobic interaction chromatography is an effective method to remove trace contaminants of inactive C1-inhibitor polymers.

Three R's of bacterial evolution: how replication, repair, and recombination frame the origin of species.

The genetic diversity of bacteria results not only from errors in DNA replication and repair but from horizontal exchange and recombination of DNA sequences from similar and disparate species as well. New individuals carrying adaptive changes are thus being spawned constantly among the population at large. When new selection pressures appear, these are the individuals that survive, at the expense of the general population, to forge new populations. Depending on the severity and uniqueness of the selection pressure, this could lead to new speciation. It is becoming more and more evident that, as nucleotide sequences of numerous loci from many bacterial strains continue to amass, horizontal transfer has played a key role in configuring the Escherichia coli chromosome. Here, we examine views, both old and new, for the role of recombination in the evolution of bacterial chromosomes. We present novel phylogenetic evidence for horizontal transfer of three genes involved in DNA replication and repair (mutS, uvrD, and polA). These data reveal a prominent role for horizontal transfer in the evolution of genes known to play a key role in the fidelity of DNA replication and, thus, ultimate survival of the organism. Our data underscore that recombination plays both a diversifying and a homogenizing role in defining the structure of the E. coli genome.

Effect of removal of duplicate isolates on cumulative susceptibility reports.

The objective of our study is to assess the impact of different methods of duplicate isolate removal on cumulative susceptibility reports. Over a 1-year period, we studied the effect of 3 methods of duplicate isolate removal on the cumulative percentage susceptibility of 9 Gram-negative bacilli to 15 antimicrobials. Raw data from which no duplicate isolates were removed (NR) were generated by the Sensititre breakpoint susceptibility testing system. D3 and D7 were methods of duplicate isolate removal defined as follows: same patient, bacterial species, irrespective of susceptibility within either three (D3) or seven (D7) calendar days of the date of the previous culture. The third method evaluated was an algorithm utilized by Cerner, a laboratory management program that defines duplicate isolates as follows: same patient, bacterial species, and NCCLS susceptibility category to an individual antimicrobial. Differences in percentage susceptibility between the three methods of duplicate isolate removal and NR were assessed. The number of isolates studied ranged from 80 (E. aerogenes) to 681 (P. aeruginosa). Of the methods of duplicate isolate removal, the highest percentage susceptibility occurred most frequently with Cerner followed by D7 and D3. Differences in percentage susceptibility between methods of removal and NR ranged from -11 to 25%, -5 to 8%, and -3 to 10%, with Cerner, D3, and D7, respectively. The percentage susceptibility was at least 5% higher than NR with a method of removal for 15 individual organism/antimicrobial combinations in which susceptibility was > or = 70% by at least one of the methods. These occurred most frequently with Enterobacter species and Cerner. Although there is no consensus on the ideal method of duplicate isolate removal, one should be cognizant that these manipulations may produce different cumulative susceptibility reports.

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains.

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.

Outbreak of Legionnaires' disease associated with a display whirlpool spa.

BACKGROUND: Recognized outbreaks of Legionnaires' disease (LD) are rare; when they occur, they provide opportunities to understand the epidemiology of the illness and improve prevention strategies. We investigated a population-based outbreak. METHODS: After the confirmation of LD in October 1996 in five people in neighbouring towns in southwest Virginia, active surveillance for additional cases was undertaken. A case-control study was conducted to identify exposures associated with illness, followed by a cohort study among employees of the facility at which the source of the outbreak was located in order to assess unrecognized exposure and illness. Samples of likely sources of LD in the facility were cultured for LEGIONELLA: RESULTS: In all, 23 laboratory-confirmed cases of LD were eventually identified. Of the 15 cases in the case-control study, 14 (93%) reported visiting a home-improvement store, compared with 12 (27%) of 45 controls (matched odds ratio [MOR] = 23.3; 95% CI : 3-182). Among home-improvement centre patrons, 10 (77%) of 13 cases questioned recalled either visiting or walking by a display whirlpool spa, compared with 3 (25%) of 12 controls (MOR = 5.5; 95% CI : 0.7-256.0). Two cases' sputum isolates were an exact match, by monoclonal antibody subtyping and arbitrarily primed polymerase chain reaction, to a whirlpool spa filter isolate from the store. Employees reporting more exposure to the display spas were more likely to report symptoms of LD or to have an elevated titre. CONCLUSIONS: This investigation shows that LD can be transmitted from a whirlpool spa used for display only, and highlights the need for minimizing the risk of transmission of LD from all water-filled spas. Key messages This paper describes an investigation of a population-based outbreak of Legionnaires' disease (LD). A case-control study first identified a home-improvement store as the likely source of the outbreak. An environmental investigation later confirmed that finding, as two cases' sputum isolates were an exact match, by monoclonal antibody subtyping and arbitrarily primed polymerase chain reaction, to a whirlpool spa filter isolate from the store. The spa was intended and used for display only.

Phylogenetic relationships of necrogenic Erwinia and Brenneria species as revealed by glyceraldehyde-3-phosphate dehydrogenase gene sequences.

Recent examination of the relationships of the dry necrosis-inducing (necrogenic) erwinias using 16S rDNA sequences demonstrated that these bacteria comprise a polyphyletic group and, therefore, have been subdivided into three distinct genera, Erwinia, Brenneria and Pectobacterium, with the classical 'amylovora' group species now being distributed nearly evenly among the first two. To further assess the molecular evolutionary relationships between current necrogenic Erwinia and Brenneria species, as well as between these genera and the exclusively soft-rotting genus Pectobacterium, the glyceraldehyde-3-phosphate dehydrogenase (gapDH) genes from 57 Erwinia and Brenneria isolates along with Pectobacterium type strains were PCR-amplified, sequenced and subjected to phylogenetic analysis. Pairwise alignments of cloned gapDH genes revealed remarkably high interspecies genetic diversity among necrogenic isolates. Four evolutionary clades of necrogenic species were described that assorted more closely to known soft-rotting species than to each other. Interclade comparisons of gapDH nucleotide sequences revealed as much genetic divergence between these four necrogenic clades as existed between necrogenic and soft-rotting clades. An examination of the phylogenetic utility of the gapDH gene in light of current 16S rDNA clustering of these species revealed varying levels of taxonomic congruence between these genes for the structure of Erwinia, Brenneria and Pectobacterium. These analyses suggest that, while gapDH possesses sufficient genetic variation to fully differentiate Erwinia and Brenneria species, the gene may not accurately reflect interspecies taxonomic relatedness among all three phytopathogenic genera.

Strengths and limitations of molecular subtyping in a community outbreak of Legionnaires' disease.

An epidemiological and microbiological investigation of a cluster of eight cases of Legionnaires' disease in Los Angeles County in November 1997 yielded conflicting results. The epidemiological part of the investigation implicated one of several mobile cooling towers used by a film studio in the centre of the outbreak area. However, water sampled from these cooling towers contained L. pneumophila serogroup 1 of another subtype than the strain that was recovered from case-patients in the outbreak. Samples from two cooling towers located downwind from all of the case-patients contained a Legionella strain that was indistinguishable from the outbreak strain by four subtyping techniques (AP-PCR, PFGE, MAb, and MLEE). It is unlikely that these cooling towers were the source of infection for all the case-patients, and they were not associated with risk of disease in the case-control study. The outbreak strain also was not distinguishable, by three subtyping techniques (AP-PCR, PFGE, and MAb), from a L. pneumophila strain that had caused an outbreak in Providence, RI, in 1993. Laboratory cross-contamination was unlikely because the initial subtyping was done in different laboratories. In this investigation, microbiology was helpful for distinguishing the outbreak cluster from unrelated cases of Legionnaires' disease occurring elsewhere. However, multiple subtyping techniques failed to distinguish environmental sources that were probably not associated with the outbreak. Persons investigating Legionnaires' disease outbreaks should be aware that microbiological subtyping does not always identify a source with absolute certainty.

Hospital characteristics associated with colonization of water systems by Legionella and risk of nosocomial legionnaires' disease: a cohort study of 15 hospitals.

OBJECTIVE: To investigate an increase in reports of legionnaires' disease by multiple hospitals in San Antonio, Texas, and to study risk factors for nosocomial transmission of legionnaires' disease and determinants for Legionella colonization of hospital hot-water systems. SETTING: The 16 largest hospitals in the cities of San Antonio, Temple, and Austin, Texas. DESIGN: Review of laboratory databases to identify patients with legionnaires' disease in the 3 years prior to the investigation and to determine the number of diagnostic tests for Legionella performed; measurement of hot-water temperature and chlorine concentration and culture of potable water for Legionella. Exact univariate calculations, Poisson regression, and linear regression were used to determine factors associated with water-system colonization and transmission of Legionella. RESULTS: Twelve cases of nosocomial legionnaires' disease were identified; eight of these occurred in 1996. The rise in cases occurred shortly after physicians started requesting Legionella urinary antigen tests. Hospitals that frequently used Legionella urinary antigen tests tended to detect more cases of legionnaires' disease. Legionella was isolated from the water systems of 11 of 12 hospitals in San Antonio; the 12th had just experienced an outbreak of legionnaires' disease and had implemented control measures. Nosocomial legionellosis cases probably occurred in 5 hospitals. The number of nosocomial legionnaires' disease cases in each hospital correlated better with the proportion of water-system sites that tested positive for Legionella (P=.07) than with the concentration of Legionella bacteria in water samples (P=.23). Hospitals in municipalities where the water treatment plant used monochloramine as a residual disinfectant (n=4) and the hospital that had implemented control measures were Legionella-free. The hot-water systems of all other hospitals (n=11) were colonized with Legionella. These were all supplied with municipal drinking water that contained free chlorine as a residual disinfectant. In these contaminated hospitals, the proportion of sites testing positive was inversely correlated with free residual chlorine concentration (P=.01). In all hospitals, hot-water temperatures were too low to inhibit Legionella growth. CONCLUSIONS: The increase in reporting of nosocomial legionnaires' disease was attributable to increased use of urinary antigen tests; prior cases may have gone unrecognized. Risk of legionnaires' disease in hospital patients was better predicted by the proportion of water-system sites testing positive for Legionella than by the measured concentration of Legionella bacteria. Use of monochloramine by municipalities for residual drinking water disinfection may help prevent legionnaires' disease.

Phylogeographic patterns of feline immunodeficiency virus genetic diversity in the domestic cat.

Feline immunodeficiency virus (FIV) has a worldwide distribution among feral and domesticated cats and in many cases induces immunodeficiency disease analogous to that of human acquired immune deficiency syndrome. FIV is genetically homologous to human immunodeficiency virus (HIV) in both genome organization and gene sequence and, like HIV, exhibits enormous sequence variation throughout the range of host species. We sampled 91 feral cats from six disparate locales and studied the phylogenetic relationships of viral DNA from infected cats using both pol and env genes (520 and 684 bp, respectively). The patterns from the two genes recapitulated previously described major FIV clades and showed concordance between phylogenetic patterns of the pol and env genes. Evidence for recombination between the pol and env genes was not found among a sampling of nine isolates, although evidence for intragenic exchange within the env gene was observed in two isolates. A small local population of cats from a rural farm in the United Kingdom had a remarkably high FIV antibody prevalence (47%), but displayed 8-fold less overall diversity of FIV genomic variation compared with FIV from different parts of the world. We interpret this low variation as a consequence of a recent monophyletic introduction of FIV into the population.