In vitro assessment of antibacterial and antiviral activity of three copper products after 200 rounds of simulated use.

Copper has well-documented antibacterial effects but few have evaluated it after prolonged use and against bacteria and viruses. Coupons from three copper formulations (solid, thermal coating, and decal applications) and carbon steel controls were subjected to 200 rounds simulated cleaning using a Wiperator™ and either an accelerated hydrogen peroxide, quaternary ammonium, or artificial sweat products. Antibacterial activity against S. aureus and P. aeruginosa was then evaluated using a modified Environmental Protection Agency protocol. Antiviral activity against coronavirus (229E) and norovirus (MNV-1) surrogates was assessed using the TCID50 method. Results were compared to untreated control coupons. One hour after inoculation, S. aureus exhibited a difference in log kill of 1.16 to 4.87 and P. aeruginosa a log kill difference of 3.39-5.23 (dependent upon copper product and disinfectant) compared to carbon steel. MNV-1 demonstrated an 87-99% reduction on each copper surfaces at 1 h and 99% reduction at 2 h compared to carbon steel. Similarly, coronavirus 229E exhibited a 97-99% reduction after 1 h and 90-99% after 2 h. Simulated use with artificial sweat did not hinder the antiviral nor the antibacterial activity of Cu surfaces. Self-sanitizing copper surfaces maintained antibacterial and antiviral activity after 200 rounds of simulated cleaning.

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids.

Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing.

Automated 16S Sequencing Using an R-Based Analysis Module for Bacterial Identification.

Sanger sequencing of the 16S rRNA gene is routinely used for the identification of bacterial isolates. However, this method is still performed mostly in more-specialized reference laboratories, and traditional protocols can be labor intensive. In this study, 99 clinical bacterial isolates were used to validate a fast, simplified, and largely automated protocol for 16S sequencing. The workflow combines real-time PCR of the first 500 bp of the bacterial 16S rRNA gene and amplicon sequencing on an automated, cartridge-based sequence analyzer. Sequence analysis, NCBI BLAST search, and result interpretation were performed using an automated R-based script. The automated workflow and R analysis described here produced results equal to those of manual sequence analysis. Of the 96 sequences with adequate quality, 90 were concordantly identified to the genus (n = 62) or species level (n = 28) compared with routine laboratory identification of the organism. One organism identification was discordant, and 5 resulted in an inconclusive identification. For sequences that gave a valid result, the overall accuracy of identification to at least the genus level was 98.9%. This simplified sequencing protocol provides a standardized approach to clinical 16S sequencing, analysis, and quality control that would be suited to frontline clinical microbiology laboratories with minimal experience. IMPORTANCE Sanger sequencing of the 16S rRNA gene is widely used as a diagnostic tool for bacterial identification, especially in cases where routine diagnostic methods fail to provide an identification, for organisms that are difficult to culture, or from specimens where cultures remain negative. Our simplified protocol is tailored toward use in frontline laboratories with little to no experience with sequencing. It provides a highly automated workflow that can deliver fast results with little hands-on time. Implementing 16S sequencing in-house saves additional time that is otherwise required to send out isolates/specimens for identification to reference laboratories. This makes results available much faster to physicians who can in turn initiate or adjust patient treatment accordingly.

Antimicrobial efficacy and durability of copper formulations over one year of hospital use.

OBJECTIVE: To evaluate 3 formulations of copper (Cu)-based self-sanitizing surfaces for antimicrobial efficacy and durability over 1 year in inpatient clinical areas and laboratories. DESIGN: Randomized control trial. SETTING: We assessed 3 copper formulations: (1) solid alloy 80% Cu-20% Ni (integral copper), (2) spray-on 80% Cu-20% Ni (spray-on) and (3) 16% composite copper-impregnated surface (CIS). In total, 480 coupons (1 cm2) of the 3 products and control surgical grade (AISI 316) stainless steel were inserted into gaskets and affixed to clinical carts used in patient care areas (including emergency and maternity units) and on microbiology laboratory bench work spaces (n = 240). The microbial burden and assessment of resistance to wear, corrosion, and material compatibility were determined every 3 months. Participants included 3 tertiary-care Canadian adult hospital and 1 pediatric-maternity hospital. RESULTS: Copper formulations used on inpatient units statistically significantly reduced bacterial bioburden compared to stainless steel at months 3 and 6. Only the integral copper product had significantly less bacteria than stainless steel at month 12. No statistically significant differences were detected in microbial burden between copper formulations and stainless-steel coupons on microbiology laboratory benches where bacterial counts were low overall. All mass changes and corrosion rates of the formulations were acceptable by engineering standards. CONCLUSIONS: Copper surfaces vary in their antimicrobial efficacy after 1 year of hospital use. Frequency of cleaning and disinfection influence the impact of copper; the greatest reduction in microbial bioburden occurred in clinical areas compared to the microbiology laboratory where cleaning and disinfection were performed multiple times daily.

Approach to Assessment of New Swabs and Viral Transport Media for SARS-CoV-2 Testing.

In light of the present pandemic of novel coronavirus disease 2019 (COVID-19) and the unprecedented high demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing worldwide, there are shortages of established specimen collection devices for respiratory viral testing for diagnostic microbiology laboratories. This creates the need to validate unverified collection devices from manufacturers that may not be a registered supplier for medical devices. As clinical laboratories do not routinely perform quality control of established collection devices, there is a need to have a systematic, robust approach to the assessment of substitute unregistered collection swabs and viral transport media (VTM). A discussion of the aspects requiring consideration when determining the suitability and implementation of new collection devices is presented. These specific assessment criteria include an inspection of device integrity, determination of swab and VTM sterility and in vitro performance, VTM stability, and examination of the clinical performance of the device. This method was used in a front-line medical microbiology laboratory on swabs and VTM from an unregistered manufacturer, with suboptimal results that precluded implementation. As the pandemic continues, it will be important for diagnostic laboratories to adopt a flexible and streamlined approach to maintaining adequate supply chains for testing reagents and materials.

Identification of New Helicobacter pylori Subpopulations in Native Americans and Mestizos From Peru.

Region-specific Helicobacter pylori subpopulations have been identified. It is proposed that the hspAmerind subpopulation is being displaced from the Americans by an hpEurope population following the conquest. Our study aimed to describe the genomes and methylomes of H. pylori isolates from distinct Peruvian communities: 23 strains collected from three groups of Native Americans (Asháninkas [ASHA, n = 9], Shimaas [SHIM, n = 5] from Amazonas, and Punos from the Andean highlands [PUNO, n = 9]) and 9 modern mestizos from Lima (LIM). Closed genomes and DNA modification calls were obtained using SMRT/PacBio sequencing. We performed evolutionary analyses and evaluated genomic/epigenomic differences among strain groups. We also evaluated human genome-wide data from 74 individuals from the selected Native communities (including the 23 H. pylori strains donors) to compare host and bacterial backgrounds. There were varying degrees of hspAmerind ancestry in all strains, ranging from 7% in LIM to 99% in SHIM. We identified three H. pylori subpopulations corresponding to each of the Native groups and a novel hspEuropePeru which evolved in the modern mestizos. The divergence of the indigenous H. pylori strains recapitulated the genetic structure of Native Americans. Phylogenetic profiling showed that Orthogroups in the indigenous strains seem to have evolved differentially toward epigenomic regulation and chromosome maintenance, whereas OGs in the modern mestizo (LIM) seem to have evolved toward virulence and adherence. The prevalence of cagA +/vacA s1i1m1 genotype was similar across populations (p = 0.32): 89% in ASHA, 67% in PUNO, 56% in LIM and 40% in SHIM. Both cagA and vacA sequences showed that LIM strains were genetically differentiated (p < 0.001) as compared to indigenous strains. We identified 642 R-M systems with 39% of the associated genes located in the core genome. We found 692 methylation motifs, including 254 population-specific sequences not previously described. In Peru, hspAmerind is not extinct, with traces found even in a heavily admixed mestizo population. Notably, our study identified three new hspAmerind subpopulations, one per Native group; and a new subpopulation among mestizos that we named hspEuropePeru. This subpopulation seems to have more virulence-related elements than hspAmerind. Purifying selection driven by variable host immune response may have shaped the evolution of Peruvian subpopulations, potentially impacting disease outcomes.

In vitro evaluation of antimicrobial efficacy and durability of three copper surfaces used in healthcare.

Antimicrobial properties of solid copper (Cu) surfaces against various microorganisms have been demonstrated, but little is known about the durability and relative antimicrobial efficacy of different Cu formulations currently used in healthcare. The aim of this study was to assess whether three different formulations of copper-bearing alloys (integral, spray-on and Cu-impregnated surfaces) and a stainless steel control differed in their antimicrobial efficacy, durability, and compatibility with hospital-grade cleaner/disinfectants. The U.S. Environmental Protection Agency draft protocol for the evaluation of bactericidal activity of Cu containing alloys was modified to more accurately reflect cleaning methods in healthcare. The three different Cu alloys were evaluated using 25 × 25 × 3 mm disks subjected to one year of simulated cleaning and disinfection using the Wiperator™ with microfiber cloths presoaked in three common hospital disinfectants: accelerated hydrogen peroxide, quaternary ammonium, or sodium hypochlorite solutions. Bactericidal activity was evaluated using Staphylococcus aureus and Pseudomonas aeruginosa. While all Cu formulations exhibited some antimicrobial activity, integral and spray-on Cu alloys showed the greatest efficacy. Assessments of durability included documentation of changes in mass, morphological changes by scanning electron microscopy, chemical composition alteration by energy-dispersive x-ray spectroscopy, and surface roughness alteration using profilometry over one year of simulated use. The integral Cu alloy had the least mass loss (0.20% and 0.19%) and abrasion-corrosion rate (6.28 and 6.09 µm/yr) compared to stainless steel. The integral product also showed the highest durability. Exposure to disinfectants affected both the antimicrobial efficacy and durability of the various copper products.

Total protein extraction and 2-D gel electrophoresis methods for Burkholderia species.

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis.

Burkholderia pseudomallei causes septicemic melioidosis with a high rate of relapse, however microbial determinants of relapse are unknown. Proteins were analyzed from sequential B. pseudomallei isolates from primary and relapsing melioidosis. Analysis by isotope tagging for relative and absolute quantitation revealed that factors required for nitric oxide detoxification (HmpA) and necessary for anaerobic growth (ArcA, ArcC and ArcB) were highly expressed in the relapse isolate. Two-dimensional gel electrophoresis revealed up-regulation of a putative hemolysin-coregulated protein in the primary isolate, and flagellin and HSP20/alpha crystalline in the relapse isolate. These observations provide targets for further analysis of latency and virulence of melioidosis.

Helicobacter pylori from Peruvian amerindians: traces of human migrations in strains from remote Amazon, and genome sequence of an Amerind strain.

BACKGROUND: The gastric pathogen Helicobacter pylori is extraordinary in its genetic diversity, the differences between strains from well-separated human populations, and the range of diseases that infection promotes. PRINCIPAL FINDINGS: Housekeeping gene sequences from H. pylori from residents of an Amerindian village in the Peruvian Amazon, Shimaa, were related to, but not intermingled with, those from Asia. This suggests descent of Shimaa strains from H. pylori that had infected the people who migrated from Asia into The Americas some 15,000+ years ago. In contrast, European type sequences predominated in strains from Amerindian Lima shantytown residents, but with some 12% Amerindian or East Asian-like admixture, which indicates displacement of ancestral purely Amerindian strains by those of hybrid or European ancestry. The genome of one Shimaa village strain, Shi470, was sequenced completely. Its SNP pattern was more Asian- than European-like genome-wide, indicating a purely Amerind ancestry. Among its unusual features were two cagA virulence genes, each distinct from those known from elsewhere; and a novel allele of gene hp0519, whose encoded protein is postulated to interact with host tissue. More generally, however, the Shi470 genome is similar in gene content and organization to those of strains from industrialized countries. CONCLUSIONS: Our data indicate that Shimaa village H. pylori descend from Asian strains brought to The Americas many millennia ago; and that Amerind strains are less fit than, and were substantially displaced by, hybrid or European strains in less isolated communities. Genome comparisons of H. pylori from Amerindian and other communities should help elucidate evolutionary forces that have shaped pathogen populations in The Americas and worldwide.

Risk factors for antibiotic-resistant Escherichia coli carriage in young children in Peru: community-based cross-sectional prevalence study.

Few studies have examined the influence of individual-, household-, and community-scale risk factors on carriage of resistant commensal bacteria. We determined children's medical, agricultural, and environmental exposures by household, pharmacy, and health facility surveys and Escherichia coli cultures of children, mothers' hands, household animals, and market chickens in Peru. Among 522 children with a positive stool culture, by log-binomial regression, using "any antibiotic" and 1-14 (versus 0) sulfa doses in the past 3 months increased children's risk, respectively, for ampicillin- and sulfamethoxazole-resistant E. coli carriage (P = 0.01-0.02). Each household member taking "any antibiotic" increased children's risk for sulfamethoxazole- and multidrug-resistant E. coli carriage (P < 0.0001). Residence in a zone where a larger proportion of households served home-raised chicken (as contrasted with intensively antibiotic-raised market chicken) protected against carrying E. coli resistant to all drugs (P = 0.0004-0.04). Environmental contamination with drug-resistant bacteria appeared to significantly contribute to children's carriage of antibiotic-resistant E. coli.

Interleukin-1 beta single-nucleotide polymorphism's C allele is associated with elevated risk of gastric cancer in Helicobacter pylori-infected Peruvians.

Particular alleles of the interleukin-1B (IL-1B) gene have been correlated with increased risk of atrophic gastritis and gastric cancer in the populations of East Asia and Europe. No such data exist from Peru, a developing country with a population genotypically different from others studied and with a high prevalence of Helicobacter pylori infection and gastric cancer. We conducted a case-control study comparing 334 hospitalized patients with atrophic gastritis or gastric cancer with 158 nonatrophic gastritis patients (controls). Conditional logistic regression analysis revealed that an increased risk of atrophic gastritis (odds ratio, 5.60) and gastric cancer (odds ratio, 2.36) was associated with the IL-1B-511 C allele. Our study is the first to establish this allele as a risk for these conditions. Given the high prevalence of H. pylori and recurrence rate after treatment, IL-1B-511 single-nucleotide polymorphism analysis may identify those individuals who would benefit most from robust H. pylori eradication efforts in Peru.

DNA-level diversity and relatedness of Helicobacter pylori strains in shantytown families in Peru and transmission in a developing-country setting.

The efficiency of transmission of a pathogen within families compared with that between unrelated persons can affect both the strategies needed to control or eradicate infection and how the pathogen evolves. In industrialized countries, most cases of transmission of the gastric pathogen Helicobacter pylori seems to be from mother to child. An alternative model, potentially applicable among the very poor in developing countries, where infection is more common and the sanitary infrastructure is often deficient, invokes frequent transmission among unrelated persons, often via environmental sources. In the present study, we compared the genotypes of H. pylori from members of shantytown households in Peru to better understand the transmission of H. pylori in developing-country settings. H. pylori cultures and/or DNAs were obtained with informed consent by the string test (a minimally invasive alternative to endoscopy) from at least one child and one parent from each of 62 families. The random amplified polymorphic DNA fingerprints of 57 of 81 (70%) child-mother strain pairs did not match, nor did the diagnostic gene sequences (>1% DNA sequence difference), independent of the child's age (range, 1 to 39 years). Most strains from siblings or other paired family members were also unrelated. These results suggest that H. pylori infections are often community acquired in the society studied. Transmission between unrelated persons should facilitate the formation of novel recombinant genotypes by interstrain DNA transfer and selection for genotypes that are well suited for individual hosts. It also implies that the effective prevention of H. pylori infection and associated gastroduodenal disease will require anti-H. pylori measures to be applied communitywide.

Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation.

We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed Helicobacter pylori infection in Peruvian shantytown residents. H. pylori isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one H. pylori strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for H. pylori strain differentiation.

Validation of string test for diagnosis of Helicobacter pylori infections.

The method of recovering Helicobacter pylori DNA or viable cells absorbed on a string that a person has swallowed and that is retrieved an hour later (string test) should be a useful alternative to traditional analysis of cells or DNA obtained by endoscopy, which is invasive, uncomfortable, relatively costly, and ill-suited for community-based and pediatric studies. Here we assayed the sensitivity and validity of the string test versus conventional endoscopic biopsy for detecting and analyzing H. pylori infection. Forty-four people with gastric complaints were studied using both H. pylori culture and urease gene (ureB) PCR. H. pylori organisms cultured from strings and biopsy specimens from the same patients were fingerprinted by the randomly amplified polymorphic DNA (RAPD) method. Biopsy sections were also hematoxylin and eosin and silver stained for H. pylori detection. H. pylori was cultured from 80% of strings and detected by PCR from 91% of strings from participants whose biopsies had been H. pylori positive by culture, PCR, and/or histology. Strains recovered from strings and biopsy specimens yielded identical or closely related RAPD profiles in each of the 24 cases tested. We conclude that the string test is a useful method for H. pylori recovery and analysis when relatively noninvasive procedures are needed.

Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

Helicobacter pylori reinfection is common in Peruvian adults after antibiotic eradication therapy.

To characterize posttreatment recurrence of Helicobacter pylori in Peru, 192 adults with H. pylori-positive gastric biopsy specimens were monitored by (14)C-Urea breath test, after eradication of H. pylori by use of amoxicillin, clarithromycin, and omeprazole. The cumulative risk of recurrence at 18 months was 30.3% (95% confidence interval, 21.4%-39.3%). Randomly amplified polymorphic DNA patterns and DNA sequence data established that, among 28 pairs of H. pylori isolates from pretreatment and recurrent infections, 6 (21%) were genetically similar, suggesting recrudescence of the previous infection, and 22 (79%) were different, suggesting reinfection with a new strain that differed from that involved in the initial infection. Eating mainly outside of the home was a risk factor for infection with a new strain (adjusted relative risk [RR], 5.07), whereas older age was a protective factor (adjusted RR, 0.20). Although an increase in the anti-H. pylori IgG antibody titer corresponded to recurrence, pretreatment and recurrent infections were similar with respect to quantitative culture colony counts and histologic characteristics, suggesting that neither prior eradication nor the memory immune response measurably alters the risk or burden of recurrent infection. Although eradication with antibiotics was successful, the high rate of reinfection suggests that treatment is unlikely to have a lasting public health effect in this setting.

[Omeprazole, amoxicillin and clarithromycin in the treatment of helicobacterpylori, in 7 and 10-day regimens]. / Tratamiento del Helicobacter pylori con omeprazol, amoxicilina y claritromicina en esquemas de 7 y 10 días.

AIM: The most accepted treatment for infection by Helicobacter pylori is the proton pump inhibitor based therapy with two antibiotics. However, there is no consensus regarding the duration. The purpose here was to compare eradication percentages in the omeprazole+amoxicillin+clarithromycin regimen administered during 7 days versus 10 days and confront the results with a previous 14-day* experience in Peru. METHOD: Patients from the Central Military Hospital and Peruvian-Japanese Hospital evidencing chronic upper gastrointestinal tract symptoms were recruited. We excluded patients with peptic ulcer. Biopsies were taken for diagnosis, for urease and PCR tests, culture and coloring with silver. Omeprazole+clarithromycin+amoxicillin was used during 7 days versus 10 days. Control endoscopy was performed one month after treatment had been completed and molecular biology techniques were used to differentiate recurrences from new infections. Susceptibility to clarithromycin was assessed. RESULTS: 36 patients were included in each group. Eradication was the same in both groups: 86.1% (31/36). In several patients in whom the bacteria persisted, the same initial nucleus was found. In a previous study* using this same regimen during 14 days, a 93% eradication was obtained. 91.18% of our samples were susceptible to clarithromycin. CONCLUSIONS: In Peru, the omeprazole+clarithromycin+amoxicillin combination gives results higher than 80% in the eradication of infection by Helicobacter pylori. The 7 and 10 days regimens eradicated the bacteria in 86% of our patients.

Cluster of type IV secretion genes in Helicobacter pylori's plasticity zone.

Some genes present in only certain strains of the genetically diverse gastric pathogen Helicobacter pylori may affect its phenotype and/or evolutionary potential. Here we describe a new 16.3-kb segment, 7 of whose 16 open reading frames are homologs of type IV secretion genes (virB4, virB7 to virB11, and virD4), the third such putative secretion gene cluster found in H. pylori. This segment, to be called tfs3, was discovered by subtractive hybridization and chromosome walking. Full-length and truncated tfs3 elements were found in 20 and 19%, respectively, of 94 strains tested, which were from Spain, Peru, India, and Japan. A tfs3 remnant (6 kb) was found in an archived stock of reference strain J99, although it was not included in this strain's published genome sequence. PCR and DNA sequence analyses indicated the following. (i) tfs3's ends are conserved. (ii) Right-end insertion occurred at one specific site in a chromosomal region that is varied in gene content and arrangement, the "plasticity zone." (iii) Left-end insertion occurred at different sites in each of nine strains studied. (iv) Sequences next to the right-end target in tfs3-free strains were absent from most strains carrying full-length tfs3 elements. These patterns suggested insertion by a transposition-like event, but one in which targets are chosen with little or no specificity at the left end and high specificity at the right end, thereby deleting the intervening DNA.