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Legionellosis is an infection acquired through inhalation of aerosolized water droplets containing Legionella bacteria. In August 2018, public health officials in New Hampshire launched an investigation into a legionellosis outbreak. They identified 49 illnesses likely associated with the outbreak and implicated an improperly maintained hot tub at a hotel. The same strain of Legionella pneumophila serogroup 1 was found in both the hot tub and in samples from two patients with Legionnaires' disease. The indoor hot tub vented to the outdoors, which is how some patients with confirmed legionellosis likely acquired the infection despite not entering the hotel during the incubation period. This outbreak is notable for 1) likely illness acquisition through the exterior vent of the hot tub room and 2) use of whole genome sequencing to link environmental and patient specimens. Collaboration among public health and environmental officials, laboratorians, and building managers was essential to determining the source of the outbreak and preventing further illness., Introduction Legionella bacteria are aerobic, Gram-negative, intracellular pathogens commonly found in fresh water and soil (Mercante & Winchell, 2015). Human infection typically is acquired through inhalation of Legionella-containing aerosols. Most [...]

Zegionella spp. are ubiquitous in nature, live in soil and water, and frequently inhabit human-made water distribution systems, hot water tanks, decorative fountains, and cooling towers (1,2). Persons with underlying [...]

Background Disseminated gonococcal infections (DGIs) are thought to be uncommon; surveillance is limited, and case reports are analyzed retrospectively or in case clusters. We describe the population-level burden of culture-confirmed DGIs through the Active Bacterial Core surveillance (ABCs) system. Methods During 2015–2016, retrospective surveillance was conducted among residents in 2 ABCs areas and prospectively in 3 ABCs areas during 2017–2019. A DGI case was defined as isolation of Neisseria gonorrhoeae from a normally sterile site. A case report form was completed for each case and antimicrobial susceptibility testing (AST) was performed on available isolates. Results During 2015–2019, 77 DGI cases were identified (a rate of 0.13 cases per 100 000 population) and accounted for 0.06% of all reported gonorrhea cases in the 3 surveillance areas. Most DGI cases were male (64%), non-Hispanic Black (68%), and ranged from 16 to 67 years of age; blood (55%) and joint (40%) were the most common sterile sites. Among 29 isolates with AST results during 2017–2019, all were susceptible to ceftriaxone. Conclusions DGI is an infrequent complication of N gonorrhoeae ; because it can quickly develop antimicrobial resistance, continued DGI surveillance, including monitoring trends in antimicrobial susceptibility, could help inform DGI treatment recommendations. [ABSTRACT FROM AUTHOR]

Disseminated gonococcal infection (DGI) is a rare complication caused by the systemic dissemination of Neisseria gonorrhoeae to normally sterile anatomical sites. Little is known about the genetic diversity of DGI gonococcal strains and how they relate to other gonococcal strains causing uncomplicated mucosal infections. We used whole genome sequencing to characterize DGI isolates (n = 30) collected from a surveillance system in Georgia, United States, during 2017–2020 to understand phylogenetic clustering among DGI as well as uncomplicated uro- and extragenital gonococcal infection (UGI) isolates (n = 110) collected in Fulton County, Georgia, during 2017–2019. We also investigated the presence or absence of genetic markers related to antimicrobial resistance (AMR) as well as surveyed the genomes for putative virulence genetic factors associated with normal human-serum (NHS) resistance that might facilitate DGI. We found that DGI strains demonstrated significant genetic variability similar to the population structure of isolates causing UGI, with sporadic incidences of geographically clustered DGI strains. DGI isolates contained various AMR markers and genetic mechanisms associated with NHS resistance. DGI isolates had a higher frequency of the porB1A allele compared with UGI (67% vs 9%, P  < .0001); however, no single NHS resistance marker was found in all DGI isolates. Continued DGI surveillance with genome-based characterization of DGI isolates is necessary to better understand specific factors that promote systemic dissemination. [ABSTRACT FROM AUTHOR]

Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of Campylobacter jejuni strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the C. jejuni F38011 wild-type strain. Complementation of the C.jejuni cbrR mutant in trans restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as cbrR (Campylobacter bile resistance regulator). While the MICs of a variety of bile salts and other detergents for the C. jejuni cbrR mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the C. jejuni cbrR mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that bile resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates resistance to bile salts in C. jejuni.

Objectives: Neisseria gonorrhoeae (gonococcus) infection is one of the most commonly reported nationally notifiable conditions in the United States. Gonococcus has developed antimicrobial resistance to each previously used antibiotic for gonorrhea therapy. However, some isolates may be still susceptible to no longer recommended, yet still effective antibiotics. This in turn suggests that targeted therapy could slow resistance development to currently recommended empirical treatments. We curated a gonococcal Ciprofloxacin Antibiotic Resistance Isolate Bank panel (Cipro-panel) as a tool for validating or developing new tests to determine ciprofloxacin susceptibility. Method: The Cipro-panel was selected using whole genome sequencing, bioinformatic tools, and antimicrobial susceptibility testing (AST) data. Isolates were further selected based on nucleotide variations in gyrA and parC genes. Results: We selected 14 unique N. gonorrhoeae isolates from the 2006–2012 Gonococcal Isolate Surveillance Project (GISP) collection. They represented a wide range of antimicrobial susceptibility to ciprofloxacin and commonly observed nucleotide variations of gyrA and parC genes. This Cipro-panel consists of 5 isolates with resistant phenotypes (MIC > = 1 μg/mL), 8 isolates with susceptible phenotypes (MIC < = 0.06 μg/mL), and 1 isolate falling in the Clinical and Laboratory Standards Institute defined intermediate range. Among the gyrA variations we observed a total of 18 SNPs. Four positions had nonsynonymous changes (nucleotide positions 272, 284, 1093, and 1783). The first two positions (272 and 284) have been linked previously with resistance to ciprofloxacin (i.e. amino acid positions 91 and 95). For the parC gene, we observed a total of 21 possible SNPs. Eight of those SNPs resulted in non-synonymous amino acid changes. One location (amino acid 87) has been previously reported to be associated with ciprofloxacin resistance. Conclusions: This Cipro-Panel is useful for researchers interested in developing clinical tests related to ciprofloxacin. It could also provide additional choices for validation, quality assurance purposes and improve antibiotic usage. [ABSTRACT FROM AUTHOR]

Neisseria gonorrhoeae multilocus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the United States. Here, we analyze a global collection of ST-9363 core-genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 core-genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 core-genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009–2011 and again during the 2012–2013 time, facilitating the clonal expansion of this core-genogroup with AZMrs in the United States. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the United States and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young core-genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers. [ABSTRACT FROM AUTHOR]

Background: Gradient strip antimicrobial susceptibility testing using Etest is conducted by local public health jurisdictions participating in the Strengthening the US Response to Resistant Gonorrhea (SURRG) program to inform public health responses to resistant gonorrhea. Proficiency testing results across the participating laboratories were analyzed and a comparison of Etest with the agar dilution method was conducted.Methods: Laboratories participating in SURRG performed Etest for azithromycin (AZM), cefixime (CFX), and ceftriaxone (CRO). Concurrence between minimum inhibitory concentrations (MICs) obtained with Etest versus the agar dilution method using corresponding isolates was defined as ±1 double dilution. Specific levels of reduced susceptibility were termed "alerts" and included isolates with the following MICs: ≥2.0 μg/mL (AZM), ≥0.25 μg/mL (CFX), and ≥0.125 μg/mL (CRO). Categorical (alert/nonalert) agreement was calculated for MICs determined using Etest and agar dilution methods.Results: Strengthening the US Response to Resistant Gonorrhea laboratories had high proficiency testing scores (≥98%) and low levels of interlaboratory variations in MICs. The overall concurrence of MICs (essential agreement) determined using agar dilution, and Etest was 96% (CRO), 96% (CFX), and 95% (AZM). Depending on the antibiotic tested, between 27% and 66% of isolates with alert MICs determined by Etest also had alert MICs using the reference agar dilution methodology; however, most of these alert MICs were detected at threshold levels.Conclusions: This study demonstrates that MICs produced by SURRG laboratories using Etest have a high level of concurrence with agar dilution. Although confirmation of specific alert MICs varied, Etest facilities rapid detection and response to emerging resistant gonorrhea. [ABSTRACT FROM AUTHOR]

Background Chlamydia trachomatis causes pelvic inflammatory disease (PID) and tubal infertility. Plasmid gene protein 3 antibody (Pgp3Ab) detects prior chlamydial infections. We evaluated for an association of high chlamydial seropositivity with sequelae using a Pgp3Ab multiplex bead array (Pgp3AbMBA). Methods We performed chlamydia Pgp3AbMBA on sera from women 18-39 years old participating in the 2013–2016 National Health and Nutrition Examination Survey (NHANES) with urine chlamydia nucleic acid amplification test results. High chlamydial seropositivity was defined as a median fluorescence intensity (MFI ≥ 50 000; low-positive was MFI > 551–<50 000. Weighted US population high-positive, low-positive, and negative Pgp3Ab chlamydia seroprevalence and 95% confidence intervals (CI) were compared for women with chlamydial infection, self-reported PID, and infertility. Results Of 2339 women aged 18–39 years, 1725 (73.7%) had sera, and 1425 were sexually experienced. Overall, 104 women had high positive Pgp3Ab (5.4% [95% CI 4.0–7.0] of US women); 407 had lowpositive Pgp3Ab (25.1% [95% CI 21.5–29.0]), and 914 had negative Pgp3Ab (69.5% [95% CI 65.5–73.4]). Among women with high Pgp3Ab, infertility prevalence was 2.0 (95% CI 1.1–3.7) times higher than among Pgp3Ab-negative women (19.6% [95% CI 10.5–31.7] versus 9.9% [95% CI 7.7–12.4]). For women with low Pgp3Ab, PID prevalence was 7.9% (95% CI 4.6–12.6) compared to 2.3% (95% CI 1.4–3.6) in negative Pgp3Ab. Conclusions High chlamydial Pgp3Ab seropositivity was associated with infertility although small sample size limited evaluation of an association of high seropositivity with PID. In infertile women, Pgp3Ab may be a marker of prior chlamydial infection. [ABSTRACT FROM AUTHOR]

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935–1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally. Resistance of Neisseria gonorrhoeae to extended spectrum cephalosporins is an increasing concern. Here, the authors conduct whole genome sequencing of isolates from the United States and find that most resistant isolates were associated with a persistent circulating lineage. [ABSTRACT FROM AUTHOR]

Abstract Background Clostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon. Results Genetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B). Conclusions These results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Abstract Background Type A1 Clostridium botulinum strains are a group of Gram-positive, spore-forming anaerobic bacteria that produce a genetically, biochemically, and biophysically indistinguishable 150 kD protein that causes botulism. The genomes of three type A1 C. botulinum strains have been sequenced and show a high degree of synteny. The purpose of this study was to characterize differences among these genomes and compare these differentiating features with two additional unsequenced strains used in previous studies. Results Several strategies were deployed in this report. First, University of Massachusetts Dartmouth laboratory Hall strain (UMASS strain) neurotoxin gene was amplified by PCR and sequenced; its sequence was aligned with the published ATCC 3502 Sanger Institute Hall strain and Allergan Hall strain neurotoxin gene regions. Sequence alignment showed that there was a synonymous single nucleotide polymorphism (SNP) in the region encoding the heavy chain between Allergan strain and ATCC 3502 and UMASS strains. Second, comparative genomic hybridization (CGH) demonstrated that the UMASS strain and a strain expected to be derived from ATCC 3502 in the Centers for Disease Control and Prevention (CDC) laboratory (ATCC 3502*) differed in gene content compared to the ATCC 3502 genome sequence published by the Sanger Institute. Third, alignment of the three sequenced C. botulinum type A1 strain genomes revealed the presence of four comparable blocks. Strains ATCC 3502 and ATCC 19397 share the same genome organization, while the organization of the blocks in strain Hall were switched. Lastly, PCR was designed to identify UMASS and ATCC 3502* strain genome organizations. The PCR results indicated that UMASS strain belonged to Hall type and ATCC 3502* strain was identical to ATCC 3502 (Sanger Institute) type. Conclusions Taken together, C. botulinum type A1 strains including Sanger Institute ATCC 3502, ATCC 3502*, ATCC 19397, Hall, Allergan, and UMASS strains demonstrate differences at the level of the neurotoxin gene sequence, in gene content, and in genome arrangement.

Analysis of the entire bont/F nucleotide sequences from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak study is reported. The results demonstrated the extensive nucleotide diversity present among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.

The entire bont/A- and bont/B-associated neurotoxin gene clusters of Clostridium botulinum type Ab strain CDC41370 and the bont/A gene of strain CDC588 was sequenced to understand the neurotoxin gene diversity among C. botulinum type Ab strains. The differences observed in the toxin gene cluster compositions and bont/A and bont/B nucleotide sequences indicated that they might have evolved from separate recombination events.

Studied were conducted on a group of five clonally related Clostridium botulinum type A strains isolated from different sources. The strains are observed to contain a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502 and also had a common toxin gene cluster composition (ha-/orfX+).

The genotype and phenotypic attributes of Campylobacter jejuni poultry isolates are examined. The abilities to invade INT 407 cells and levels of secretion of virulence proteins are found to be different in both isolates.

Legionella spp. are the cause of a severe bacterial pneumonia known as Legionnaires’ disease (LD). In some cases, current genetic subtyping methods cannot resolve LD outbreaks caused by common, potentially endemic L. pneumophila (Lp) sequence types (ST), which complicates laboratory investigations and environmental source attribution. In the United States (US), ST1 is the most prevalent clinical and environmental Lp sequence type. In order to characterize the ST1 population, we sequenced 289 outbreak and non-outbreak associated clinical and environmental ST1 and ST1-variant Lp strains from the US and, together with international isolate sequences, explored their genetic and geographic diversity. The ST1 population was highly conserved at the nucleotide level; 98% of core nucleotide positions were invariant and environmental isolates unassociated with human disease (n = 99) contained ~65% more nucleotide diversity compared to clinical-sporadic (n = 139) or outbreak-associated (n = 28) ST1 subgroups. The accessory pangenome of environmental isolates was also ~30–60% larger than other subgroups and was enriched for transposition and conjugative transfer-associated elements. Up to ~10% of US ST1 genetic variation could be explained by geographic origin, but considerable genetic conservation existed among strains isolated from geographically distant states and from different decades. These findings provide new insight into the ST1 population structure and establish a foundation for interpreting genetic relationships among ST1 strains; these data may also inform future analyses for improved outbreak investigations. [ABSTRACT FROM AUTHOR]

Cooling towers (CTs) are a leading source of outbreaks of Legionnaires’ disease (LD), a severe form of pneumonia caused by inhalation of aerosols containing Legionella bacteria. Accordingly, proper maintenance of CTs is vital for the prevention of LD. The aim of this study was to determine the distribution of Legionella in a subset of regionally diverse US CTs and characterize the associated microbial communities. Between July and September of 2016, we obtained aliquots from water samples collected for routine Legionella testing from 196 CTs located in eight of the nine continental US climate regions. After screening for Legionella by PCR, positive samples were cultured and the resulting Legionella isolates were further characterized. Overall, 84% (164) were PCR-positive, including samples from every region studied. Of the PCR-positive samples, Legionella spp were isolated from 47% (78), L. pneumophila was isolated from 32% (53), and L. pneumophila serogroup 1 (Lp1) was isolated from 24% (40). Overall, 144 unique Legionella isolates were identified; 53% (76) of these were Legionella pneumophila. Of the 76 L. pneumophila isolates, 51% (39) were Lp1. Legionella were isolated from CTs in seven of the eight US regions examined. 16S rRNA amplicon sequencing was used to compare the bacterial communities of CT waters with and without detectable Legionella as well as the microbiomes of waters from different climate regions. Interestingly, the microbial communities were homogenous across climate regions. When a subset of seven CTs sampled in April and July were compared, there was no association with changes in corresponding CT microbiomes over time in the samples that became culture-positive for Legionella. Legionella species and Lp1 were detected frequently among the samples examined in this first large-scale study of Legionella in US CTs. Our findings highlight that, under the right conditions, there is the potential for CT-related LD outbreaks to occur throughout the US. [ABSTRACT FROM AUTHOR]

Legionella pneumophila was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires’ disease (LD) at a Pennsylvania veterans’ convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of Legionella epidemiology and scientific research. Only one of the original isolates, “Philadelphia-1”, has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with L. pneumophila isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with L. pneumophila str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of “Broad Street pneumonia”, and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for Legionella outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set. [ABSTRACT FROM AUTHOR]

A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations ("outbreaks") that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila. This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. [ABSTRACT FROM AUTHOR]

Background: In the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/ A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains. Results: The genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain. Conclusions: This study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds. [ABSTRACT FROM AUTHOR]

The article discusses cases of N. gonorrhoeae with the penA60 allele in the southern Nevada. Topics include that Southern Nevada Health District (SNHD) provided Centers for Disease Control & Prevention with all N. gonorrhoeae NAAT-positive specimens from all clinics; and the importance of surveillance programs like Gonococcal Isolate Surveillance Project (GISP) as effective tools for identification of antimicrobial-resistant pathogens.

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bout/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multiocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260pg/mL and 21pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water. [Copyright &y& Elsevier]

Background: Type A1 Clostridium botulinum strains are a group of Gram-positive, spore-forming anaerobic bacteria that produce a genetically, biochemically, and biophysically indistinguishable 150 kD protein that causes botulism. The genomes of three type A1 C. botulinum strains have been sequenced and show a high degree of synteny. The purpose of this study was to characterize differences among these genomes and compare these differentiating features with two additional unsequenced strains used in previous studies. Results: Several strategies were deployed in this report. First, University of Massachusetts Dartmouth laboratory Hall strain (UMASS strain) neurotoxin gene was amplified by PCR and sequenced; its sequence was aligned with the published ATCC 3502 Sanger Institute Hall strain and Allergan Hall strain neurotoxin gene regions. Sequence alignment showed that there was a synonymous single nucleotide polymorphism (SNP) in the region encoding the heavy chain between Allergan strain and ATCC 3502 and UMASS strains. Second, comparative genomic hybridization (CGH) demonstrated that the UMASS strain and a strain expected to be derived from ATCC 3502 in the Centers for Disease Control and Prevention (CDC) laboratory (ATCC 3502*) differed in gene content compared to the ATCC 3502 genome sequence published by the Sanger Institute. Third, alignment of the three sequenced C. botulinum type A1 strain genomes revealed the presence of four comparable blocks. Strains ATCC 3502 and ATCC 19397 share the same genome organization, while the organization of the blocks in strain Hall were switched. Lastly, PCR was designed to identify UMASS and ATCC 3502* strain genome organizations. The PCR results indicated that UMASS strain belonged to Hall type and ATCC 3502* strain was identical to ATCC 3502 (Sanger Institute) type. Conclusions: Taken together, C. botulinum type A1 strains including Sanger Institute ATCC 3502, ATCC 3502*, ATCC 19397, Hall, Allergan, and UMASS strains demonstrate differences at the level of the neurotoxin gene sequence, in gene content, and in genome arrangement. [ABSTRACT FROM AUTHOR]