Non-fermenting Gram-negative Rods Research Articles

A rapid and inexpensive protocol to screen for third generation cephalosporin-resistant and non-fermenting Gram-negative rods directly in positive blood cultures

Third generation cephalosporins are frequently used in the first-line treatment of Gram-negative rod (GNR) bacteremia but are unsuitable in the case of extended-spectrum-beta-lactamase-producing Enterobacterales (ESBL-E) or non-fermenting GNR infections. The aim of this study was to develop and evaluate a simple and rapid two-test protocol involving oxidase and β-Lacta tests performed directly on positive blood culture broth as a preliminary screen for non-fermenting or third generation cephalosporins-resistant GNR. The diagnostic performance of this approach was evaluated on 294 bottles for the oxidase test and 267 bottles for the β-Lacta Test. The sensitivity and specificity of the oxidase test were respectively 93.1% and 100%, and the sensitivity of the β-Lacta Test for ESBL-E was 100% and the specificity 99.5%. This simple protocol, which can be implemented in all laboratories and performed in only 20 min, may be a valuable tool to optimize first-line antibiotic therapy for bacteremia.

Clinical performance of ASTA SepsiPrep kit in direct bacterial identification and antimicrobial susceptibility test using MicroIDSys Elite and VITEK-2 system.

BackgroundRapid and accurate microbial identification and antimicrobial susceptibility testing (AST) are essential for timely use of appropriate antimicrobial agents for bloodstream infection. To shorten the time for isolating colonies from the positive blood culture, various preparation methods for direct identification using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) system were developed. Here, we evaluated the SepsiPrep kit (ASTA Corp.) for direct identification of microorganisms and AST from positive blood cultures using MicroIDSys Elite MALDI‐TOF MS system (ASTA Corp.) and VITEK‐2 system (bioMérieux).MethodsFor direct identification, a total of 124 prospective monomicrobial positive blood culture bottles were included. For direct identification, the pellet was prepared by centrifugation and washing twice. For direct AST, the pellet was suspended in 0.45% saline and adjusted to McFarland 0.5. The results from the direct identification and AST using MicroIDSys Elite and VITEK‐2 system were compared to those from the conventional method performed with pure colony subcultured on agar plate.ResultsCompared to the conventional method using pure colony, correct direct identification rate was 96.5% and 98.5% for 57 gram‐positive isolates and 67 gram‐negative isolates, respectively. For direct AST, among the 55 gram‐positive isolates, the categorical agreement (CA) for staphylococci, streptococci, and enterococci was 96.7%, 98.4%, and 94.1%, respectively. For 66 gram‐negative isolates, the CA for Enterobacterales and non‐fermentative gram‐negative rods was 99.0% and 96.6%, respectively.ConclusionsThe SepsiPrep kit was easy to use combined with MicroIDSys Elite and VITEK‐2 system and also, the correct identification and AST rate were very high.

Мониторинг ведущей грамотрицательной микрофлоры и антибиотикорезистентности при остеомиелите

Introduction There is an urgent need for a surveillance system of regular monitoring of specific bacteria inducing various types of osteomyelitis to identify resistant isolates and optimize the use of antibiotics. Objective: monitoring of specific gram-negative bacteria and analysis of the antibiotic resistance of the strains isolated from osteomyelitis patients over a three-year period. Results and discussion P. aeruginosa was the first most common pathogen among gram-negative microorganisms isolated from the patients between 2017 and 2019. Prevalence of the isolates identified in 2019 decreased by 9.6 % as compared to 2017. Next frequently encountered clinical isolates were Enterobacter sp., Acinetobacter sp., Klebsiella sp. There was a twofold increase in K. pneumoniae strains isolated in 2019. Analysis of antibiotic susceptibility testing data revealed multiresistance of the Acinetobacter sp. strains in 2019 despite the total decrease in resistant isolates in 2017 and 2018. Among non-fermenting gram-negative rods, the species being resistant to imipenem were shown to increase by 5.4 times. Overall antibiotic resistance was on rise. Increased antimicrobial resistance to beta-lactam antibiotics also combined with BLaC inhibitors was observed in Enterobacteriaceae population. Meropenem was found to be effective against most bacteria with growing drug resistance observed as compared with recent years. The antibiotic resistance profiles of Klebsiella sp. strains appeared to be high at antimicrobial testing. Conclusion Diverse bacterial morphology of gram-negative species and increasing proportion of drug-resistant strains isolated in osteomyelitis cases have necessitated regular monitoring of multiresistant clinical isolates for adjustment of empirical antibiotic therapies.

Evaluation of a polymyxin drop test for polymyxin resistance detection among non-fermentative gram-negative rods and enterobacterales resistant to carbapenems.

To assess the performance of the drop test for polymyxin B resistance detection among Enterobacterales and non-fermentative gram-negative rods resistant to carbapenems. Seven hundred and fifteen carbapenem-resistant isolates were tested: 628 Enterobacterales species and 87 non-fermentative gram-negative rods. For the polymyxin drop test, concentrations range from 0.25 to 8.0µg/mL. Broth microdilution, as gold standard, was performed using in-house-prepared panels and interpreted according to the CLSI guidelines. Results were interpreted in terms of categorical agreements and discrepancies. Accuracy for a drop of polymyxin B at 2.0, 4.0 and 8.0 was calculated as better cutoff for resistance determination. No very major error was observed among all isolates, and 95.5% of agreement was observed among Enterobacterales, particularly for Klebsiella pneumoniae. A higher accuracy (95.1%) was obtained when a single drop of polymyxin B at 4.0μg/mL was applied. Polymyxin drop test presented >95% of categorical agreement, without very major errors, for KPC-producing K.pneumoniae isolates. An accuracy of 95.1% was obtained with a single drop at 4.0μg/mL polymyxin B. Polymyxin B drop is an easy and feasible test and may allow a reduction on the turnaround time for polymyxin resistance detection and impacting on early implementation of accurate therapeutic interventions.

Performance of the BD Phoenix CPO detect assay for detection and classification of carbapenemase-producing organisms

Increasing worldwide, prevalence of carbapenem-resistant gram-negative bacteria demands urgent a need for rapid detection and accurate identification of carbapenemases. The BD Phoenix CPO detect (PCD) assay possesses an in-built capacity for parallel susceptibility testing and detection of carbapenemases. Here, the ability of the assay to detect and classify carbapenemase production was tested in a collection of carbapenem-resistant Enterobacterales and non-fermentative gram-negative rods. The ability of the PCD assay to detect and classify carbapenemases was investigated in a collection of 194 clinical, carbapenem-resistant isolates (Enterobacterales [n = 65]; non-fermentative gram-negative rods [n = 129]). AST results were compared to MICS determined by gradient diffusion to determine accuracy of the PCD assay. The accuracy of the PCD assay to detect carbapenemases was compared to the results of molecular isolate characterization using a LDT multiplex carbapenemase PCR assay. All 194 isolates classified as carbapenem-resistant by reference susceptibility testing were also classified correctly as CRO by the PCD assay. Performance analysis of the PCD assay to detect carbapenemase production revealed an overall sensitivity of 98.29% and specificity of 17.95% for the detection of carbapenemase production. For the classification of carbapenemases classes A, B, and D, the PCD correctly classified 79.17% Enterobacterales and 67.16% non-fermentative gram-negative rods. The PCD assay is a reliable tool for the detection of carbapenem resistance and allows for parallel analysis of carbapenemase production. However, while sensitivity is high, low specificity in carbapenemase detection and erroneous classification demands mandatory confirmation by alternative methods, especially in non-fermentative gram-negative bacteria.

Multimodal strategy in surgical site infections control and prevention in orthopaedic patients \u2013 a 10-year retrospective observational study at a Polish hospital

IntroductionSurgical site infections (SSIs) are among the most common healthcare-associated infections. They are associated with longer post-operative hospital stays, additional surgical procedures, risk of treatment in intensive care units and higher mortality.Material and methodsSSIs were detected in patients hospitalized in a 40-bed orthopaedics ward in 2009–2018. The total number of study patients was 15,678. The results were divided into two 5-year periods before and after the introduction of the SSI prevention plan. The study was conducted as part of a national Healthcare-Associated Infections Surveillance Programme, following the methodology recommended by the HAI-Net, European Centre for Disease Prevention and Control Program (ECDC).ResultsOne hundred sixty eight SSIs were detected in total, including 163 deep SSIs (SSI-D). The total SSI incidence rate was 1.1%, but in hip prosthesis: 1.2%, in knee prosthesis: 1.3%, for open reduction of fracture (FX): 1.3%, for close reduction of fracture (CR): 1.5, and 0.8% for other procedures. 64% of SSI-D cases required rehospitalisation. A significantly reduction in incidence was found only after fracture reductions: FX and CR, respectively 2.1% vs. 0.7% (OR 3.1 95%CI 1.4–6.6, p < 0.01) and 2.1 vs. 0.8% (OR 2.4 95%CI 1.0–5.9, p < 0.05). SSI-Ds were usually caused by Gram-positive cocci, specially Staphylococcus aureus, 74 (45.7%); Enterobacteriaceae bacillis accounted for 14.1% and Gram-negative non-fermenting rods for 8.5%.ConclusionsThe implemented SSI prevention plan demonstrated a significant decrease from 2.1 to 0.7% in SSI-D incidence only in fracture reductions, without changes in epidemiology SSI incidence rates in other procedures. Depending on the epidemiological situation in the ward, it is worthwhile to surveillance of SSIs associated to different types of orthopaedic surgery to assess the risks of SSI and take preventive measures.

The applicability of gene sequencing and MALDI-TOF to identify less common gram-negative rods (Advenella, Castellaniella, Kaistia, Pusillimonas and Sphingobacterium) from environmental isolates.

Our aim was to identify less common non-fermenting gram-negative rods during the bioremediation process. Five genera were found: Advenella, Castellaniella, Kaistia, Pusillimonas and Sphingobacterium, for a total of 15 isolates. Therefore, we evaluated the applicability of four methods currently available for bacteria identification: (1) conventional biochemical methods, (2) the VITEK®-2 system, (3) MALDI-TOF mass spectrometry and (4) 16S rRNA gene sequencing. The biochemical methods and the VITEK®-2 system were reliable only for the Sphingobacterium isolate and solely at the genus level. Both MALDI-TOF mass spectrometry platforms (Bruker and VITEK® MS) did not achieve reliable identification results for any of these genera. 16S rRNA gene sequencing identified eight isolates to the species level but not to the subspecies level, when applicable. The remaining seven isolates were reliably identified through 16S rRNA gene sequencing to the genus level only. Our findings suggest that the detection and identification of less common genera (and species) that appeared at certain moments during the bioremediation process can be a challenge to microbiologists considering the most used techniques. In addition, more studies are required to confirm our results.

Effect of Inhibition of Bacterial Proliferation by Neutral Electrolytic Water inDental Unit Waterlines

Background: Microbial contamination in dental unit waterlines (DUWLs) has recently become an important issue in the field of dental infection control. Using neutral electrolytic water as a new disinfecting method has attracted considerable attention. However, long term data about the effect of using neutral electrolytic water in clinical settings are scarce. This is the first study to evaluate the long term effectiveness of inhibition of bacterial proliferation using the purification system to supply neutral electrolytic water to refine the waterworks in DUWLs. Methods: Before the study, we investigated the actual levels of bacterial contamination in DUWLs. Then we did thorough cleaning of DUWLs and water samples were collected 6 dental units. Three dental units assigned as Group A had purification systems that used neutral electrolytic water, and the other 3 units were the Control group. Water samples were collected from the gargle water, high-speed handpieces and the three-way syringe. We utilized the equipment in Group A, and both groups were maintained for daily clinic work for 14 months. We counted the bacterial colony forming units (cfu) for each sample and identified the pathogenic bacterial species. Results: 3 and 14 months later, no microbes were detected during the study period in Group A whereas numbers of cfu which grew from the Control group increased and glucose non-fermenting gram-negative rod of possible pathogenic organisms to human were identified in the control groups. Conclusions: The water purification system using neutral electrolytic water was effective to control the proliferation of bacteria and could maintain a hygienic environment in DUWLs.

Evaluation of the Alfred AST® system for rapid antimicrobial susceptibility testing directly from positive blood cultures

To assess the concordance of antimicrobial susceptibility testing results obtained by the Alfred AST® system performed directly from positive blood cultures in comparison with the standard susceptibility test results performed from isolated colonies by an automated broth microdilution method and to determine the applicability of Alfred AST® system in the routine of our blood culture laboratory. This system is based on the detection of growth by turbidimetry through a technology based on light scattering. Antimicrobial susceptibility testing was performed directly from positive bottles by the Alfred AST® system (Alifax, Padova, Italy). The broth microdilution method (MicroScan, Beckman Coulter, CA, USA) performed to the isolates was considered the standard for comparison. We evaluated 115 significant episodes of bacteremia produced by 51 Gram-negative Enterobacterales, 8 Pseudomonas spp., 2 non-fermenting Gram-negative rods, 7 Staphylococcus aureus, 23 coagulase-negative Staphylococcus, 12 Enterococcus spp., and 12 Streptococcus spp. We performed 828 susceptibility determinations with a categorical agreement with the standard method of 97.1%. Only 24 errors (2.9%) were detected. It should be pointed out that for staphylococci and glycopeptides the correlation was only 87% and for non-fermenting Gram-negative rods and piperacillin/tazobactam was only 88.9%. Time to get antibiogram results by Alfred AST® system was 5 versus 48h for the standard microdilution method from the isolated colonies. The Alfred AST® system is a useful and rapid method to obtain antimicrobial susceptibility results within the same work shift after blood culture positivity.

Treatment of purulent-inflammatory complications in a combat gunshot trauma

Purpose of the study. Justification of the effective treatment tactics of the wounded with the gunshot fractures of the long bones.&#x0D; Materials and methods. The examination and treatment of 123 wounded with gunshot fractures were performed. All wounded were divided into 3 groups according to clinical observation. For the first clinical group the traditional treatment of the combat fractures with osteometallosynthesis out of injury zone was done. In the second group, treatment was added with negative pressure therapy. In the third clinical group Wounded got a VAC-associated therapy with the proposed of counter-drainage of wounds with the flow-washing irrigation with antiseptic solutions Decasan + 3% hydrogen peroxide. A bacteriological study of the wounds` exudates was carried out, and the sensitivity of microorganisms to antibiotics was determined.&#x0D; Results. The assay of microbiological investigation of the wounds of patients, which got explosive and mineexplosive injuries, demonstrated a predominance gramnegative microflora in the wound microbiocenoses such as Acinetobacter spp. (53% of cases) and Pseudomonas spp. (15% of cases). Gram-positive cocci were isolated from 22,2% of cases. The analysis of the antibiotic sensitivity data of gram-negative nonfermentative rods showed a high level of resistance to most antibacterial. All strains of acinetobacteria and pseudomonads were susceptible to polymyxin B and colistin, but resistant to unprotected and protected aminopenicillins (amoxicillin/clavulanate, ampicillin/ sulbactam). Acceleration of regenerative processes in the wound under the influence of VAC-therapy (formation of healthy granulations, disappearance of edema) in patients with negative pressure suppression (II HS, III HS) led to a reduction of hospitalization period, which took in average 7,8 ± 1,2 days, that was 5,2 ± 0,8 days less than in a control group. In the third group of wounded, a mixture of Decasan and 3% hydrogen peroxide in the proportion of 3 : 1 was used for rinsing of wounds. On the third day tissue edema decreased in 94,45% of the wounded in that observation group, while in the second clinical group it was observed in 88,89% of patients. The duration of the hydration phase in the wound process was reduced to 5,7 days. The period of complete healing of the wounds was shorter for 2,5 days. The period of indoor stay of the wounded of this group in the hospital decreased from 14,97 to 10,8 days.&#x0D; Conclusions. Prevalence of gram-negative microorganisms in a gunshot wound and their high degree of resistance to antibiotics should be noticed when one takes a decision about empirical antibiotic therapy in the wounded. Observed clinical results of the proposed negative pressure therapy with counter-drainage of wounds by setting of flow-washing irrigation with a mixture of antiseptics Decasan and 3% hydrogen peroxide allow recommending this scheme for treatment of wounded with gunshot fractures of long bones.

Occurrence of Antibiotic-Resistant Bacteria in Therapy Pools and Surrounding Surfaces.

The number of patients colonized with antibiotic-resistant bacteria is increasing in health care facilities. Because transmission of antibiotic-resistant bacteria is feared, there exist reports that the affected patients are frequently excluded from hydrotherapy, which is a non-invasive and beneficial treatment used for patients with different diseases. Data from the literature suggest that deficient water disinfection measures exist, which are not always sufficient to kill all released bacteria. If the pool water is not disinfected properly, it may also infect the bathers. Immunocompromised patients are particularly susceptible to be infected with (antibiotic-resistant) bacteria. In order to determine the distribution of antibiotic-resistant bacteria in the pool water treatment system and the pool environment and to estimate the associated transmission risk we analyzed samples from eleven health care facilities. Antibiotic-resistant bacteria were found in the water and surface samples collected. One hundred and two antibiotic-resistant isolates from water samples and 307 isolates from surrounding surfaces were obtained, respectively. The majority of the isolates belonged to non-fermenting Gram-negative rods, like Pseudomonas spp. Some isolates were resistant to a wide range of the tested antibiotics. The results indicate a relation between the number of isolates in water samples and the number of patients using the pools in combination with deficiencies in water treatment. In the pool environment the highest number of isolates was obtained from barefoot areas and floor cleaning equipment.

Achromobacter xylosoxidans Bacteremia in a Child with Neutropenia

Achromobacter xylosoxidans is a non-fermentative, aerobic, oxidase, and catalase-positive Gram-negative rod similar to Pseudomonas species. This organism colonizes aquatic environments and can cause nosocomial infections, especially in patients with immune deficiency such as human immunodeficiency virus infection, cancer, cystic fibrosis, neutropenia, and immunoglobulin M deficiency. Infections are found as bacteremia, pneumonia, meningitis, urinary tract infection, abscess formation, and osteomyelitis. It is known that most effective antibiotics are piperacillin/tazobactam, meropenem, and trimethoprim/sulfamethoxazol. But there is no optimal antibiotic therapy so far. We present a case of Achromobacter xylosoxidans bacteremia in a 13-month-old Korean girl who had past history of neutropenia. Key words: Achromobacter xylosoxidans; Bacteremia; Neutropenia; Child

Case Report: First report of Elizabethkingia miricola infection in a patient with cystic fibrosis

Elizabethkingia miricola is a rare non-fermenting Gram-negative rod that has previously been reported to be associated with blood stream and pulmonary abscess infections, but never before in cystic fibrosis (CF). Here we present the first reported case of Elizabethkingia miricola infection in a patient with CF and discuss the management options. We describe a patient with CF in whom we observed clinical and spirometric evidence of pulmonary exacerbation with the associated growth of E. miricola in sputum culture. The period of clinical instability was observed to coincide with the obtainment of four sputum samples from which E. miricola was cultured; improvement was seen following treatment with ciprofloxacin and the subsequent eradication of E. miricola. We conclude that E. miricola is able to survive in the CF lung and in this case was associated with pulmonary exacerbation. Empirical treatment with fluoroquinolones is appropriate, based on our experience.

Mycobacterium chimaera in heater-cooler units used during cardiac surgery – growth and decontamination

Background: Previous studies have identified patients infected with Mycobacterium chimaera (M. chimaera) subsequent to cardiac surgery. Water tanks in heater-cooler units (HCUs) used cardiac heart surgery was traced as source. The aim was to investigate occurrence of M. chimaera and other microorganisms in HCUs and evaluate the silver-ion cleaning routine.Method: Five HCUs were disinfected with silver-ions and examined for mycobacteria directly (15 min) after the disinfection procedures and later on three occasions (3, 6, 10 weeks). One HCU was selected for additional investigation of the presence of other microorganisms. In addition, tap water from five sinks in the surgical department was examined for the presence of mycobacteria and other microorganisms.Results: M. chimaera grew in all the HCU water tanks and in 35 of the 40 HCU samples. Three of the samples also contained Mycobacterium gordonae. When the selected HCU tanks were analysed directly after the disinfection procedure bacteria and fungi were found but no non-fermenting Gram-negative rods. These HCU samples contained a doubled to 3 fold amount of bacteria compared to initial tap water samples. No mycobacteria were found in any sample from the five water taps.Conclusion: The silver-ion cleaning routine was insufficient and M. chimaera was found in all HCUs. However, no mycobacteria were found in any sample from the five water taps suggesting another source of colonization. It is probable that residual water and biofilm are of importance. Our results emphasize the need for improved disinfection procedures and improved construction of the HCUs.

Case Report: First report of Elizabethkingia miricola infection in a patient with cystic fibrosis.

Elizabethkingia miricola is a rare non-fermenting Gram-negative rod that has previously been reported to be associated with blood stream and pulmonary abscess infections, but never before in cystic fibrosis (CF). Here we present the first reported case of Elizabethkingia miricola infection in a patient with CF and discuss the management options. We describe a patient with CF in whom we observed clinical and spirometric evidence of pulmonary exacerbation with the associated growth of E. miricola in sputum culture. The period of clinical instability was observed to coincide with the obtainment of four sputum samples from which E. miricola was cultured; improvement was seen following treatment with ciprofloxacin and the subsequent eradication of E. miricola. We conclude that E. miricola is able to survive in the CF lung and in this case was associated with pulmonary exacerbation. Empirical treatment with fluoroquinolones is appropriate, based on our experience.

Epidemiology of healthcare-associated infections in Polish intensive care. A multicenter study based on active surveillance.

The aim of this study was to evaluate the incidence of health care-associated infections (HAIs) in patients treated in Polish intensive care units (ICUs). This retrospective analysis was based on the results of active targeted surveillance, according to the recommendation of the ECDC (European Centre of Disease Control and Prevention, HAI-Net light protocol), conducted in 2013-2015 in seven ICUs for adults located in southern Poland (observational study). The incidence of HAI was 22.6% and 28.7/1000 person-days (pds). The incidence of pneumonia (PN) was 8.0%, bloodstream infections (BSIs) 7.2% and urinary tract infections (UTIs) 3.7%. The incidence per 1000 pds was as follows: PN 10.2, BSIs 9.2 and UTIs 4.7. PN was the most common source of secondary bloodstream infection (45%); the second was UTIs (22%). Mortality (directly and indirectly) associated with HAI was 10.8% and was related to the presence of PN or primary BSIs. HAIs were usually (69.2%) caused by Gram-negative bacteria; Klebsiella spp. and nonfermenting Gram-negative rods demonstrated very high antibiotic resistance. Despite the lack of widely implemented active targeted surveillance programmes and top-down incentives, it is possible to carry out effective surveillance of HAIs in ICUs in Poland. The results of this study are comparable with the ECDC data, but the results are alarmingly high in two fields: epidemiology of PN and BSIs and very high antibiotic resistance in Gram-negative rods, which indicate the need for intense control in this area and for further studies to clarify the source of the observed discrepancy.

Sphingobacterium spiritivorum bacteremia due to cellulitis in an elderly man with chronic obstructive pulmonary disease and congestive heart failure: a case report

BackgroundSphingobacterium spiritivorum is a glucose non-fermenting Gram-negative rod, formerly classified as one of the Flavobacterium species. It is characterized by a large number of cellular membrane sphingophospholipids. Sphingobacterium species are ubiquitous and isolated from natural environments, such as soil and water. However, they rarely cause infections in humans. Only a limited number of cases have been reported in elderly and immunocompromised patients with underlying diseases and predisposing factors.Case presentationAn 80-year-old Japanese man with chronic obstructive pulmonary disease and congestive heart failure visited the Kariya Toyota General Hospital, Aichi, Japan with the chief complaint of fever accompanied by chills and left leg pain. At initial presentation, he was distressed and dyspneic. He was febrile (38.8 °C), and his left foot was swollen with reddening and tenderness. We diagnosed him as having cellulitis, and he was hospitalized for antibiotic therapy. Initially, he was treated with intravenously administered cefazolin, but after the isolation of a glucose non-fermenting Gram-negative rod from blood cultures, we decided to switch cefazolin to intravenously administered meropenem on day 4, considering the antibiotic resistance of the causative organism. The causative organism was identified as S. spiritivorum on day 6. His condition gradually stabilized after admission. Meropenem was switched to orally administered levofloxacin on day 12. He was discharged on day 16 and treated successfully without any complications.ConclusionsAlthough S. spiritivorum is rare, with limited cases isolated from cellulitis, it should be considered as a causative organism in elderly and immunocompromised patients with cellulitis. Blood cultures are the key to correct diagnosis and appropriate treatment.

Diversity of Ochrobactrum species in food animals, antibiotic resistance phenotypes and polymorphisms in the blaOCH gene.

Twenty-six lactose non-fermenting, oxidase, urease and citrate-positive Gram-negative rods, isolated from broiler chickens, pigs and cattle at slaughter, were subjected to the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rDNA sequencing for identification. Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Ochrobactrum isolates resistant to third-generation cephalosporins were PCR-screened for the presence of the Ochrobactrum anthropi ampC gene (blaOCH). A 547-bp internal segment of blaOCH in the Ochrobactrum spp isolates was amplified with a newly designed primer set, and a phylogenetic reconstruction based on the complete amino acid sequence of blaOCH obtained from nine Ochrobactrum strains in our collection and 20 O. anthropi available in the GenBank was undertaken. All the Ochrobactrum isolates were resistant to the expanded-spectrum beta-lactams and streptomycin. None of the isolates was resistant to imipenem while 41.7% to 50.0% of them were resistant to fluoroquinolones. The blaOCH gene was detected in 16 (66.7%) and 20 (83.3%) of the 24 Ochrobactrum isolates (O. intermedium/O. tritici species), using primers designed for O. anthropi and the newly designed primer set, respectively. Six blaOCH variants grouped into two divergent clusters were identified. This is the first report of the complete nucleotide sequence of the blaOCH gene in non-antropi Ochrobactrum species.

Sanitary evaluation of domestic water supply facilities with storage tanks and detection of Aeromonas, enteric and related bacteria in domestic water facilities in Okinawa Prefecture of Japan

To provide for temporary restrictions of the public water supply system, storage tanks are commonly installed in the domestic water systems of houses and apartment buildings in Okinawa Prefecture of Japan. To learn more about the sanitary condition and management of these water supply facilities with storage tanks (hereafter called “storage tank water systems”) and the extent of bacterial contamination of water from these facilities, we investigated their usage and the existence of Aeromonas, enteric and related bacteria.Verbal interviews concerning the use and management of the storage tank water systems were carried out in each randomly sampled household. A total of 54 water samples were collected for bacteriological and physicochemical examinations. Conventional methods were used for total viable count, fecal coliforms, identification of bacteria such as Aeromonas, Enterobacteriaceae and non-fermentative Gram-negative rods (NF-GNR), and measurement of residual chlorine. On Aeromonas species, tests for putative virulence factor and an identification using 16S rRNA and rpoB genes were also performed.Water from the water storage systems was reported to be consumed directly without boiling in 22 of the 54 houses (40.7%). 31 of the sampled houses had installed water storage tanks of more than 1 cubic meter (m3) per inhabitant, and in 21 of the sampled houses, the tank had never been cleaned. In all samples, the total viable count and fecal coliforms did not exceed quality levels prescribed by Japanese waterworks law. Although the quantity of bacteria detected was not high, 23 NF-GNR, 14 Enterobacteriaceae and 5 Aeromonas were isolated in 42.6%, 7.4% and 3.7% of samples respectively. One isolated A. hydrophila and four A. caviae possessed various putative virulence factors, especially A. hydrophila which had diverse putative pathogenic genes such as aer, hlyA, act, alt, ast, ser, and dam. Many bacteria were isolated when the concentration of residual chlorine was below 0.1 mg/l and the water temperature was above 20 °C. These results suggest that elevated water temperature and mismatch between tank size and water demand lead to loss of residual chlorine in tap water. Therefore, to minimize growth of aquatic bacteria such as Aeromonas spp. and Pseudomonas spp., we recommend that an appropriate size tank and/or volume of stored water is always used, and also suggest installation of some means of reducing water temperature such as shading.

Multiresistant opportunistic pathogenic bacteria isolated from polluted rivers and first detection of nontuberculous mycobacteria in the Algerian aquatic environment

Opportunistic infections constitute a major challenge for modern medicine mainly because the involved bacteria are usually multiresistant to antibiotics. Most of these bacteria possess remarkable ability to adapt to various ecosystems, including those exposed to anthropogenic activities. This study isolated and identified 21 multiresistant opportunistic bacteria from two polluted rivers, located in Algiers. Cadmium, lead, and copper concentrations were determined for both water samples to evaluate heavy metal pollution. High prevalence of Enterobacteria and non-fermentative Gram-negative rods was found and a nontuberculous Mycobacterium (NTM) strain was isolated. To the best of our knowledge, this is the first detection of NTM in the Algerian environment. The strains were tested for their resistance against 34 antibiotics and 8 heavy metals. Multiple antibiotics and heavy metals resistance was observed in all isolates. The two most resistant strains, identified as Acinetobacter sp. and Citrobacter freundii, were submitted to plasmid curing to determine if resistance genes were plasmid or chromosome encoded. Citrobacter freundii strain P18 showed a high molecular weight plasmid which seems to code for resistance to zinc, lead, and tetracycline, at the same time. These findings strongly suggest that anthropized environments constitute a reservoir for multiresistant opportunistic bacteria and for circulating resistance genes.