The importance of effect sizes when comparing cycle threshold values of SARS-CoV-2 variants.

PURPOSE: We aimed to elaborate whether cycle threshold (Ct) values differ significantly between wild type SARS-CoV-2 (wtV) and certain viral variants and how strong or weak a potential significant effect might be. METHODS: In a retrospective study, we investigated 1873 SARS-CoV-2 positive samples for the occurrence of viral marker mutations. Age, gender, clinical setting, days after onset of symptoms, and Ct values were recorded. Statistical analysis was carried out with special consideration of effect sizes. RESULTS: During the study period wtV was detected in 1013 samples (54%), while 845 (45%) patients carried the Alpha variant of concern (VOC), and 15 (1%) the Beta VOC. For further analysis, only wtV and the Alpha VOC were included. In a multi-factor ANOVA and post-hoc test with Bonferroni-correction for the age groups we found significant main-effects for Ct values of the viral variant (wtV mean 26.4 (SD 4.27); Alpha VOC mean 25.0 (SD 3.84); F (1,1850) = 55.841; p < .001) and the clinical setting (outpatients: mean 25.7 (SD 4.1); inpatients: mean 27.0 (SD 4.2); F (1,1850) = 8.520, p = .004). However, since the effect sizes were very small (eta squared for the Alpha VOC = .029 and the clinical setting = .004), there was only a slight trend towards higher viral loads of the Alpha VOC compared to wtV. CONCLUSIONS: In order to compare different variants of SARS-CoV-2 the calculation of effect sizes seems to be necessary. A combination of p-values as estimates of the existance of an effect and effect sizes as estimates of the magnitude of a potential effect may allow a better insight into transmission mechanisms of SARS-CoV-2.

Where have the enteric viruses gone? - Differential effects on frequent causes of infectious diarrhoea by SARS-CoV-2 pandemic lockdown measures.

BACKGROUND: Measures of distancing, wearing face/medical masks and lockdown introduced in many countries to meet the challenges of the SARS-CoV-2 pandemic have led to gross changes in the epidemiology of important infections. The observation of decline of positive norovirus tests after introduction of lockdown in Germany led us to investigate changes in the detection of major causes of diarrhoea by comparing pre-pandemic quarters (PPQ: 1Q/17 through 1Q/20) since 2017 and pandemic quarters (PQ: 2Q/20 through 1Q/21). METHODS AND SETTING: Bioscientia Laboratory Ingelheim is a large regional clinical pathology laboratory serving > 50 hospitals and > 5000 general practitioners and specialist outpatient practices located in the federal states Hesse, Rhineland-Palatinate and North Rhine-Westphalia, Germany. Antigen detection assays were used for detection of astrovirus, adenovirus, rotavirus, and Campylobacter antigen and Clostridium difficile Toxin A/B, while norovirus was detected by qualitative RT-PCR. FINDINGS: The mean positivity-ratios of norovirus, adenovirus and astrovirus assays were 3-20 fold lower in periods PQ (2Q/20 through 1Q/21) compared to PPQ (1Q/17 through 1Q/20) (p<.01). The mean positivity-ratio was lower in PQ compared to PPQ for rotavirus (p=.31), but failed to reach statistical significance, while for campylobacter antigen (p=.91) and C. difficile Toxin A/B (p=.17) the mean positivity-ratio was even higher in PQ compared to PPQ. CONCLUSIONS: Apparently, hygienic measures used to contain the SARS-CoV-2 pandemic have differential effects on incidence of diarrhoea viruses as compared to bacterial gastrointestinal agents, particularly C. difficile, which may lead to re-evaluate measures implemented against this important cause of nosocomial diarrhoea.

Changing epidemiology of vancomycin-resistant Enterococcus faecium: Results of a genome-based study at a regional neurological acute hospital with intensive care and early rehabilitation treatment.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) are an emerging threat worldwide. In Germany, a VRE-belt with higher VREfm prevalences transversing its central east-west axis and including the state of Hesse was previously described. Recently, we detected a predominant VREfm clone in hospitals throughout the Rhine-Main metropolitan area of Hesse. AIM: Here we expanded our study on VREfm to a regional neurological acute hospital outside of the metropolitan area with patient referrals from throughout Hesse and the neighboring federal state of Rhineland-Palatinate. MATERIAL/METHODS: VREfm isolates obtained between 2016-2018 from a regional neurological acute hospital with intensive care and early rehabilitation units were investigated (n=55). Patient data was collected and analyzed together with whole-genome sequencing data to investigate antibiotic resistance and virulence determinants of the VREfm. The population structure of VREfm was investigated using the Core genome-based multilocus sequence typing (cgMLST). FINDINGS: The average age of the patients was 67.1 years. For 96% of the patients, a previous hospital stay was reported. 64% of the patients were treated with antibiotics. All VREfm harbored the vanB vancomycin resistance gene. The multilocus sequence types (STs) detected changed abruptly from four different STs in 2016 to a predominant ST in 2017 and 2018 (ST117). Most of the ST117 isolates were members of the cgMLST type CT71. CONCLUSION: The results indicate a sudden shift of the VREfm population structure from a semi-heterogeneous population to a pre-dominant clone within an interval of two years. Further investigations are warranted to understand the epidemiology and emergence of this clone.

Prevalence of SARS-CoV-2 IgG antibodies in a large prospective cohort study of elite football players in Germany (May-June 2020): implications for a testing protocol in asymptomatic individuals and estimation of the rate of undetected cases.

OBJECTIVES: Elite professional football players and staff are a unique group that might give insight into the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in Germany and thus can serve as a model for geographical distribution and an estimation of undetected infections. METHODS: In this prospective cohort study seroprevalence was determined twice in May and June 2020 in players and staff from the German Bundesliga. As screening assays, a commercial ELISA (Euroimmun) and a chemiluminescent immunoassay (CLIA) (Roche) were used, and an in-house neutralization assay (NT) was used as reference standard. Participants were tested twice weekly using PCR from nasopharyngeal and/or oropharyngeal swabs. RESULTS: Seroprevalence (NT used as confirmation) in 2164 samples from 1184 players and staff was rather similar in May (23/1157, 1.99%) and June (21/1007, 2.09%). All participants were PCR-negative during the study period. Significant regional differences in seroprevalence were not observed. When comparing seroprevalence with the cumulative incidence of infections derived from the German notification system (subgroup matching to cohort; men, age 20-69 years), IgG was found eight to ten times more frequently, pointing to a high rate of undetected infection. ELISA and CLIA correlated only moderately (κ 0.52). CONCLUSIONS: Seroprevalence with a high-quality diagnostic in Germany seemed to be around 2%. The number of undetected infections seems to be eight to ten times higher than in notification data. The quality of antibody assays is rather variable, thus results should ideally be confirmed at least by a second assay to prove IgG positivity.

Successful return to professional men's football (soccer) competition after the COVID-19 shutdown: a cohort study in the German Bundesliga.

OBJECTIVES: To evaluate the restart of the German Bundesliga (football (soccer)) during the COVID-19 pandemic from a medical perspective. METHODS: Participants were male professional football players from the two highest German leagues and the officials working closely with them. Our report covers nine match days spread over 9 weeks (May to July 2020). Daily symptom monitoring, PCR testing for SARS-CoV-2 RNA twice weekly, and antibody tests (on two occasions-early during the phase in May 2020 and in the week of the last match) were conducted. Target variables were: (1) onset of typical COVID-19 symptoms, (2) positive PCR results, and (3) IgG seroconversion against SARS-CoV-2. All detected seroconversions were controlled by neutralisation tests. FINDINGS: Suspicious symptoms were reported for one player; an immediate additional PCR test as well as all subsequent diagnostic and antibody tests proved negative for coronavirus. Of 1702 regularly tested individuals (1079 players, 623 officials members), 8 players and 4 officials tested positive during one of the first rounds of PCR testing prior to the onset of team training, 2 players during the third round. No further positive results occurred during the remainder of the season. 694 players and 291 officials provided two serum samples for antibody testing. Nine players converted from negative/borderline to positive (without symptoms); two players who initially tested positive tested negative at the end of the season. 22 players remained seropositive throughout the season. None of the seroconversions was confirmed in the neutralisation test. CONCLUSION: Professional football training and matches can be carried out safely during the COVID-19 pandemic. This requires strict hygiene measures including regular PCR testing.

Contrasting effects of linezolid on healthy and dysfunctional human neutrophils: reducing C5a-induced injury.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of ventilator-associated pneumonia (VAP). Patients with VAP have poorly functioning neutrophils, related to increased levels of the complement fragment C5a. The antibiotic linezolid has been useful in controlling MRSA-related VAP infections; however clinical benefit does not always correlate with antimicrobial effect, suggesting the possibility of immunomodulatory properties. Here the effects of linezolid on healthy and dysfunctional neutrophils (modelled by C5a-induced injury) was investigated. Functional assays (killing, phagocytosis, transmigration, and respiratory burst) were used to assess the effects of pre-, co- and post-incubating linezolid (0.4-40 mg/L) with healthy neutrophils relative to those with C5a-induced injury. C5a decreased neutrophil killing, and phagocytosis of MRSA. Furthermore, C5a significantly decreased neutrophil transmigration to IL-8, but did not affect respiratory burst. Co-incubation of linezolid significantly improved killing of MRSA by dysfunctional neutrophils, which was supported by concomitant increases in phagocytosis. Conversely linezolid impaired killing responses in healthy neutrophils. Pre- or post-incubation of linezolid prior or following C5a induced injury had no effect on neutrophil function. This study suggests that linezolid has immunomodulatory properties that protect human neutrophils from injury and provides insight into its mode of action beyond a basic antibiotic.

Temporal Changes in Patient-Matched Staphylococcus epidermidis Isolates from Infections: towards Defining a 'True' Persistent Infection.

Staphylococcus epidermidis is found naturally on the skin but is a common cause of persistent orthopaedic device-related infections (ODRIs). This study used a pan-genome and gene-by-gene approach to analyse the clonality of whole genome sequences (WGS) of 115 S. epidermidis isolates from 55 patients with persistent ODRIs. Analysis of the 522 gene core genome revealed that the isolates clustered into three clades, and MLST analysis showed that 83% of the isolates belonged to clonal complex 2 (CC2). Analysis also found 13 isolate pairs had different MLST types and less than 70% similarity within the genes; hence, these were defined as re-infection by a different S. epidermidis strain. Comparison of allelic diversity in the remaining 102 isolates (49 patients) revealed that 6 patients had microevolved infections (>7 allele differences), and only 37 patients (77 isolates) had a 'true' persistent infection. Analysis of the core genomes of isolate pairs from 37 patients found 110/841 genes had variations; mainly in metabolism associated genes. The accessory genome consisted of 2936 genes; with an average size of 1515 genes. To conclude, this study demonstrates the advantage of using WGS for identifying the accuracy of a persistent infection diagnosis. Hence, persistent infections can be defined as 'true' persistent infections if the core genome of paired isolates has ≤7 allele differences; microevolved persistent infection if the paired isolates have >7 allele differences but same MLST type; and polyclonal if they are the same species but a different MLST type.

Evaluation of a novel immunochromatographic lateral flow assay for rapid detection of OXA-48, NDM, KPC and VIM carbapenemases in multidrug-resistant Enterobacteriaceae.

PURPOSE: The global spread and increasing clinical impact of carbapenemase-producing bacteria are alarming. Rapid diagnostic techniques for carbapenemase detection are of the utmost importance to prevent delays in efficient antibiotic therapy and the control of spread in hospitals. Recently, multiplex immunochromatographic lateral flow tests (ICTs) for the fast detection of carbapenemase-producers have become commercially available. We evaluated a novel multiplex ICT for the rapid detection of OXA-48, KPC, NDM and VIM carbapenemases. METHODOLOGY: One hundred well-characterized multidrug-resistant Enterobacteriaceae were analysed by RESIST-4 (Coris, BioConcept, Gembloux, Belgium). The reference standard included confirmation at the molecular level at the German National Reference Laboratory for multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany).Results/Key findings. The tested ICT was highly reliable, showing 100 % sensitivity and 100 % specificity. CONCLUSION: As it is fast, easy to perform and has few technical requirements, the ICT represents a good opportunity to improve turnaround times and patient care for the clinical microbiology laboratory.

Disease-associated genotypes of the commensal skin bacterium Staphylococcus epidermidis.

Some of the most common infectious diseases are caused by bacteria that naturally colonise humans asymptomatically. Combating these opportunistic pathogens requires an understanding of the traits that differentiate infecting strains from harmless relatives. Staphylococcus epidermidis is carried asymptomatically on the skin and mucous membranes of virtually all humans but is a major cause of nosocomial infection associated with invasive procedures. Here we address the underlying evolutionary mechanisms of opportunistic pathogenicity by combining pangenome-wide association studies and laboratory microbiology to compare S. epidermidis from bloodstream and wound infections and asymptomatic carriage. We identify 61 genes containing infection-associated genetic elements (k-mers) that correlate with in vitro variation in known pathogenicity traits (biofilm formation, cell toxicity, interleukin-8 production, methicillin resistance). Horizontal gene transfer spreads these elements, allowing divergent clones to cause infection. Finally, Random Forest model prediction of disease status (carriage vs. infection) identifies pathogenicity elements in 415 S. epidermidis isolates with 80% accuracy, demonstrating the potential for identifying risk genotypes pre-operatively.

Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany). 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

Limitations in the use of PSMγ, agr, RNAIII, and biofilm formation as biomarkers to define invasive Staphylococcus epidermidis from chronic biomedical device-associated infections.

Staphylococcus epidermidis is a common cause of biomedical device-associated infections. Agr is the major quorum sensing system in staphylococci and regulates virulence factors. Four agr-specificity groups exist in S. epidermidis, and chronic S. epidermidis infections are hypothesised to select for agr-negative phenotypes. Therefore, we investigated S. epidermidis strains from prosthetic joint- and catheter-associated infections to establish i) whether an infection selects for an agr-negative phenotype; ii) the importance of PSMγ and iii) if the agr-specificity group is infection dependent. S. epidermidis nasal isolates from healthy volunteers were used as controls. The distribution of agr-specificity groups was significantly different between infection and control episodes, but did not distinguish between the infection types. PSMγ secretion was used to determine agr-activity and HPLC analysis showed that 44% of prosthetic and 32% of catheter-associated episodes produced no PSMγ in comparison to 8% of the control strains. However, PSMγ expression did not always correlate with RNAIII up-regulation, indicating that PSMγ synthesis is likely influenced by additional post-transcriptional control. The data suggests chronic S. epidermidis infections favour agr-specificity group 1 but the results suggest that they do not select for an agr-negative phenotype. Further studies are required to explore the mechanisms underlying the selection and survival of these S. epidermidis phenotypes isolated from biomedical device-associated infections.

Complete Genome Sequence of Staphylococcus epidermidis 1457.

Staphylococcus epidermidis 1457 is a frequently utilized strain that is amenable to genetic manipulation and has been widely used for biofilm-related research. We report here the whole-genome sequence of this strain, which encodes 2,277 protein-coding genes and 81 RNAs within its 2.4-Mb genome and plasmid.

Recombination-Mediated Host Adaptation by Avian Staphylococcus aureus.

Staphylococcus aureus are globally disseminated among farmed chickens causing skeletal muscle infections, dermatitis, and septicaemia. The emergence of poultry-associated lineages has involved zoonotic transmission from humans to chickens but questions remain about the specific adaptations that promote proliferation of chicken pathogens. We characterized genetic variation in a population of genome-sequenced S. aureus isolates of poultry and human origin. Genealogical analysis identified a dominant poultry-associated sequence cluster within the CC5 clonal complex. Poultry and human CC5 isolates were significantly distinct from each other and more recombination events were detected in the poultry isolates. We identified 44 recombination events in 33 genes along the branch extending to the poultry-specific CC5 cluster, and 47 genes were found more often in CC5 poultry isolates compared with those from humans. Many of these gene sequences were common in chicken isolates from other clonal complexes suggesting horizontal gene transfer among poultry associated lineages. Consistent with functional predictions for putative poultry-associated genes, poultry isolates showed enhanced growth at 42 °C and greater erythrocyte lysis on chicken blood agar in comparison with human isolates. By combining phenotype information with evolutionary analyses of staphylococcal genomes, we provide evidence of adaptation, following a human-to-poultry host transition. This has important implications for the emergence and dissemination of new pathogenic clones associated with modern agriculture.

Biofilm Morphotypes and Population Structure among Staphylococcus epidermidis from Commensal and Clinical Samples.

Bacterial species comprise related genotypes that can display divergent phenotypes with important clinical implications. Staphylococcus epidermidis is a common cause of nosocomial infections and, critical to its pathogenesis, is its ability to adhere and form biofilms on surfaces, thereby moderating the effect of the host's immune response and antibiotics. Commensal S. epidermidis populations are thought to differ from those associated with disease in factors involved in adhesion and biofilm accumulation. We quantified the differences in biofilm formation in 98 S. epidermidis isolates from various sources, and investigated population structure based on ribosomal multilocus typing (rMLST) and the presence/absence of genes involved in adhesion and biofilm formation. All isolates were able to adhere and form biofilms in in vitro growth assays and confocal microscopy allowed classification into 5 biofilm morphotypes based on their thickness, biovolume and roughness. Phylogenetic reconstruction grouped isolates into three separate clades, with the isolates in the main disease associated clade displaying diversity in morphotype. Of the biofilm morphology characteristics, only biofilm thickness had a significant association with clade distribution. The distribution of some known adhesion-associated genes (aap and sesE) among isolates showed a significant association with the species clonal frame. These data challenge the assumption that biofilm-associated genes, such as those on the ica operon, are genetic markers for less invasive S. epidermidis isolates, and suggest that phenotypic characteristics, such as adhesion and biofilm formation, are not fixed by clonal descent but are influenced by the presence of various genes that are mobile among lineages.

Complicated sea urchin-induced wound infection caused by Vibrioalginolyticus and Staphylococcus lugdunensis in a 14-year-old boy.

INTRODUCTION: Wound infections with Vibrio alginolyticus, a Gram-negative bacterium found in all temperate oceans, are rarely reported. However, a rising incidence of wound infections caused by V. alginolyticus requires better knowledge about this infectious agent. CASE PRESENTATION: We report the case of a 14-year-old boy suffering from a wound infection caused by V. alginolyticus and Staphylococcus lugdunensis after stepping on a sea urchin. Despite wound debridement and antibiotic therapy with cefaclor, the lesion did not heal over several weeks. After identification of the pathogens and antibiotic-susceptibility testing, antibiotic therapy was switched to ciprofloxacin, followed by trimethoprim/sulfamethoxazole. Two months after the accident the wound was re-epithelialized. Follow up after 6 months revealed a painful scar. CONCLUSION: Non-cholera vibrios like V. alginolyticus should be considered as possible causative agents in seawater-contaminated wounds. S. lugdunensis is a relevant pathogen in mixed wound infections. Early microbiological diagnosis and antibiotic-susceptibility testing is crucial to prevent therapeutic failure.

Effects of polysaccharide intercellular adhesin (PIA) in an ex vivo model of whole blood killing and in prosthetic joint infection (PJI): A role for C5a.

BACKGROUND: A major complication of using medical devices is the development of biofilm-associated infection caused by Staphylococcus epidermidis where polysaccharide intercellular adhesin (PIA) is a major mechanism of biofilm accumulation. PIA affects innate and humoral immunity in isolated cells and animal models. Few studies have examined these effects in prosthetic joint infection (PJI). METHODS: This study used ex vivo whole blood modelling in controls together with matched-serum and staphylococcal isolates from patients with PJI. RESULTS: Whole blood killing of PIA positive S. epidermidis and its isogenic negative mutant was identical. Differences were unmasked in immunosuppressed whole blood pre-treated with dexamethasone where PIA positive bacteria showed a more resistant phenotype. PIA expression was identified in three unique patterns associated with bacteria and leukocytes, implicating a soluble form of PIA. Purified PIA reduced whole blood killing while increasing C5a levels. In clinically relevant staphylococcal isolates and serum samples from PJI patients; firstly complement C5a was increased 3-fold compared to controls; secondly, the C5a levels were significantly higher in serum from PJI patients whose isolates preferentially formed PIA-associated biofilms. CONCLUSIONS: These data demonstrate for the first time that the biological effects of PIA are mediated through C5a in patients with PJI.

Ecological Overlap and Horizontal Gene Transfer in Staphylococcus aureus and Staphylococcus epidermidis.

The opportunistic pathogens Staphylococcus aureus and Staphylococcus epidermidis represent major causes of severe nosocomial infection, and are associated with high levels of mortality and morbidity worldwide. These species are both common commensals on the human skin and in the nasal pharynx, but are genetically distinct, differing at 24% average nucleotide divergence in 1,478 core genes. To better understand the genome dynamics of these ecologically similar staphylococcal species, we carried out a comparative analysis of 324 S. aureus and S. epidermidis genomes, including 83 novel S. epidermidis sequences. A reference pan-genome approach and whole genome multilocus-sequence typing revealed that around half of the genome was shared between the species. Based on a BratNextGen analysis, homologous recombination was found to have impacted on 40% of the core genes in S. epidermidis, but on only 24% of the core genes in S. aureus. Homologous recombination between the species is rare, with a maximum of nine gene alleles shared between any two S. epidermidis and S. aureus isolates. In contrast, there was considerable interspecies admixture of mobile elements, in particular genes associated with the SaPIn1 pathogenicity island, metal detoxification, and the methicillin-resistance island SCCmec. Our data and analysis provide a context for considering the nature of recombinational boundaries between S. aureus and S. epidermidis and, the selective forces that influence realized recombination between these species.

Structural basis of Staphylococcus epidermidis biofilm formation: mechanisms and molecular interactions.

Staphylococcus epidermidis is a usually harmless commensal bacterium highly abundant on the human skin. Under defined predisposing conditions, most importantly implantation of a medical device, S. epidermidis, however, can switch from a colonizing to an invasive life style. The emergence of S. epidermidis as an opportunistic pathogen is closely linked to the biofilm forming capability of the species. During the past decades, tremendous advance regarding our understanding of molecular mechanisms contributing to surface colonization has been made, and detailed information is available for several factors active during the primary attachment, accumulative or dispersal phase of biofilm formation. A picture evolved in which distinct factors, though appearing to be redundantly organized, take over specific and exclusive functions during biofilm development. In this review, these mechanisms are described in molecular detail, with a highlight on recent insights into multi-functional S. epidermidis cell surface proteins contributing to surface adherence and intercellular adhesion. The integration of distinct biofilm-promoting factors into regulatory networks is summarized, with an emphasis on mechanism that could allow S. epidermidis to flexibly adapt to changing environmental conditions present during colonizing or invasive life-styles.

The Yersinia pestis†HmsCDE regulatory system is essential for blockage of the oriental rat flea (Xenopsylla cheopis), a classic plague vector.

The second messenger molecule cyclic diguanylate is essential for Yersinia pestis biofilm formation that is important for blockage-dependent plague transmission from fleas to mammals. Two diguanylate cyclases (DGCs) HmsT and Y3730 (HmsD) are responsible for biofilm formation in vitro and biofilm-dependent blockage in the oriental rat flea Xenopsylla cheopis respectively. Here, we have identified a tripartite signalling system encoded by the y3729-y3731 operon that is responsible for regulation of biofilm formation in different environments. We present genetic evidence that a putative inner membrane-anchored protein with a large periplasmic domain Y3729 (HmsC) inhibits HmsD DGC activity in vitro while an outer membrane Pal-like putative lipoprotein Y3731 (HmsE) counteracts HmsC to activate HmsD in the gut of X. cheopis. We propose that HmsE is a critical element in the transduction of environmental signal(s) required for HmsD-dependent biofilm formation.