Practice of standardization of CLSI M45 A3 antimicrobial susceptibility testing of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in Guangdong Province 2017-2021.

The concentration of antimicrobial agents in environments like water and food has increased rapidly, which led to a rapid increase in antimicrobial resistance levels in the environment. Monitoring of bacterial resistance levels is considered as a necessary means to control the bacterial resistance. Reference standards are critical for antimicrobial susceptibility testing. CLSI M45 A3 standard defines pathogenic microorganisms that cause infections less frequently than those covered by CLSI M02, M07, and M100 as Infrequently Isolated or Fastidious Bacteria and specifies antimicrobial susceptibility testing methods. Our study investigated the epidemiology and antimicrobial susceptibility testing data of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in 70 hospitals in Guangdong Province between 2017 and 2021. We defined testing methods other than those specified in CLSI M45 A3 as "Non-Standardized." The proportion of standardized antimicrobial susceptibility testing for penicillin increased significantly (Corynebacterium spp. 17.4% vs. 50.0% p < 0.05; Micrococcus spp. 50.0% vs. 77.8% p < 0.05; Abiotrophia spp. and Granulicatella spp. 21.4% vs. 90.9% p < 0.001), while for cefotaxime (Corynebacterium spp. 0.0% vs. 45.2% p < 0.05; Abiotrophia spp. and Granulicatella spp. 0.0% vs. 14.3% p = 0.515) and vancomycin increased finitely. Non-standardized methods were used for all other antimicrobials. Due to limitations in the economic and medical environment, some clinical laboratories are unable to fully comply with CLSI M45 A3 standard. We recommend that CLSI should add breakpoints for disk diffusion method to improve the standardization of antimicrobial susceptibility testing.

Antibiotic Prescription Patterns for Acute Respiratory Infections in Rural Primary Healthcare Settings in Guangdong, China: Analysis of 162,742 Outpatient Prescriptions.

Overuse and inappropriate use of antibiotics are important contributors to bacterial antimicrobial resistance (AMR), especially in ambulatory primary healthcare (PHC) settings in low- and middle-income countries. This study aimed to investigate antibiotic prescription patterns among patients with acute respiratory infections (ARIs) in rural PHC facilities in the Guangdong Province, China. A total of 444,979 outpatient prescriptions were extracted from the electronic medical record system of 35 township health centers (THCs) and 2 community health centers (CHCs) between November 2017 and October 2018. We used the chi-square test to analyze the antibiotic prescription patterns and binary logistic regression to explore patient-related factors associated with antibiotic prescriptions. Of the 162,742 ARI prescriptions, 85.57% (n = 139,259) included at least one antibiotic. Among the 139,259 prescriptions with antibiotics, 37.82% (n = 52,666) included two or more antibiotics, 55.29% (n = 76,993) included parenteral antibiotics, and 56.62% (n = 78,852) included Watch group antibiotics. The binary logistic regression indicated that (1) female patients were slightly less likely to be prescribed antibiotics than males (adjusted odds ratio (OR) = 0.954, 95% confidence interval [CI] [0.928-0.981]; p = 0.001); and (2) compared to patients aged ≤5 years, those who were 6-15 years old (adjusted OR = 1.907, 95% CI [1.840-1.978]; p < 0.001), 16-60 years old (adjusted OR = 1.849, 95% CI [1.785-1.916]; p < 0.001), and >60 years old (adjusted OR = 1.915, 95% CI [1.810-2.026]; p < 0.001) were more likely to be prescribed antibiotics. The overuse and irrational use of antibiotics in PHC settings remain major healthcare challenges in rural Guangdong. Thus, it is imperative to implement targeted antimicrobial stewardship (AMS) policies to address this problem.

Adaptive Evolution Compensated for the Plasmid Fitness Costs Brought by Specific Genetic Conflicts.

New Delhi metallo-ß-lactamase (NDM)-carrying IncX3 plasmids is important in the transmission of carbapenem resistance in Escherichia coli. Fitness costs related to plasmid carriage are expected to limit gene exchange; however, the causes of these fitness costs are poorly understood. Compensatory mutations are believed to ameliorate plasmid fitness costs and enable the plasmid's wide spread, suggesting that such costs are caused by specific plasmid-host genetic conflicts. By combining conjugation tests and experimental evolution with comparative genetic analysis, we showed here that the fitness costs related to ndm/IncX3 plasmids in E. coli C600 are caused by co-mutations of multiple host chromosomal genes related to sugar metabolism and cell membrane function. Adaptive evolution revealed that mutations in genes associated with oxidative stress, nucleotide and short-chain fatty acid metabolism, and cell membranes ameliorated the costs associated with plasmid carriage. Specific genetic conflicts associated with the ndm/IncX3 plasmid in E. coli C600 involve metabolism and cell-membrane-related genes, which could be ameliorated by compensatory mutations. Collectively, our findings could explain the wide spread of IncX3 plasmids in bacterial genomes, despite their potential cost.

In Vitro Activity of Various Sulbactam Compounds and Piperacillin/Tazobactam against Clinical Isolates of Different Gram-Negative Bacteria.

To provide direction for clinical application and pharmaceutical exploitation, the in vitro activity of sulbactam compounds and PIP/TAZ 8 : 1 against clinical isolates of Gram-negative bacteria (GNB, n = 976) was evaluated according to Clinical and Laboratory Standards Institute (CLSI) 2019. By minimal inhibitory concentrations (MICs), the resistance rate of all GNB to AMP/SBT 2 : 1 (56.9-100%) was significantly higher than other drugs, except the resistance rate of Acinetobacter baumannii (Aba, n = 204) to piperacillin/tazobactam (PIP/TAZ 8 : 1, 78.4%) which was close to it (76.5%). Additionally, the resistance rate of Aba to other compounds except AMP/SBT 2 : 1 differed greatly, but that of Klebsiella pneumonia (Kpn, n = 205) varied rarely. In addition, Escherichia coli (Eco, n = 204) and Kpn demonstrated low and high resistance rates, respectively. Compared with cefoperazone/sulbactam (CPZ/SBT 2 : 1), PIP/TAZ 8 : 1 had advantage in anti-Eco (RR = 0.5and OR = 2.17) and anti-Kpn activity (RR = 0.88and OR = 1.27), while its activity against Pseudomonas aeruginosa (Pae: n = 194, RR = 0.91, and OR = 1.12), Aba (RR = 1.31 and OR = 0.41), and other Enterobacteriaceae (other Ebc: n = 169, RR = 1.40, and OR = 0.62) was not better than CPZ/SBT 2 : 1. Although it had advantage against Eco (RR = 0.60 and OR = 1.78), Pae (RR = 0.67 and OR = 1.63), and Aba (RR = 0.70 and OR = 2.05), the inhibition effect of piperacillin/sulbactam (PIP/SBT 2 : 1) against Kpn (RR = 0.94 and OR = 1.12) and other Ebc was just similar with CPZ/SBT 2 : 1 (RR = 0.93 and OR = 1.10). Furthermore, the anti-Eco (RR = 0.70 and OR = 1.50), anti-Kpn (RR = 0.89 and OR = 1.24), and anti-Pae (RR = 0.74 and OR = 1.46) activities of ceftazidime/sulbactam (CAZ/SBT 1 : 1) had a weak advantage, while its activity against Aba (RR = 0.94 and OR = 1.15) and other Ebc (RR = 0.79 and OR = 1.36) was just close to CPZ/SBT 2 : 1. Moreover, the inhibitory effect of PIP/SBT 1 : 1 against all tested clinical species was more active than CPZ/SBT 2 : 1, while that of CAZ/SBT 2 : 1 against all species of bacteria analyzed was weaker than the controls.

Mutations in porin LamB contribute to ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae.

Ceftazidime-avibactam (CAZ-AVI) shows promising activity against carbapenem-resistant Klebsiella pneumoniae (CRKP), however, CAZ-AVI resistance have emerged recently. Mutations in KPCs, porins OmpK35 and/or OmpK36, and PBPs are known to contribute to the resistance to CAZ-AVI in CRKP. To identify novel CAZ-AVI resistance mechanism, we generated 10 CAZ-AVI-resistant strains from 14 CAZ-AVI susceptible KPC-producing K. pneumoniae (KPC-Kp) strains through in vitro multipassage resistance selection using low concentrations of CAZ-AVI. Comparative genomic analysis for the original and derived mutants identified CAZ-AVI resistance-associated mutations in KPCs, PBP3 (encoded by ftsI), and LamB, an outer membrane maltoporin. CAZ-AVI susceptible KPC-Kp strains became resistant when complemented with mutated blaKPC genes. Complementation experiments also showed that a plasmid borne copy of wild-type lamB or ftsI gene reduced the MIC value of CAZ-AVI in the induced resistant strains. In addition, blaKPC expression level increased in four of the six CAZ-AVI-resistant strains without KPC mutations, indicating a probable association between increased blaKPC expression and increased resistance in these strains. In conclusion, we here identified a novel mechanism of CAZ-AVI resistance associated with mutations in porin LamB in KPC-Kp.

An Emerging Clone (ST457) of Acinetobacter baumannii Clonal Complex 92 With Enhanced Virulence and Increasing Endemicity in South China.

Background: The global dissemination of carbapenem-resistant Acinetobacter baumannii clonal complex (CC) 92 has become an urgent public health concern. Methods: A. baumannii isolates were collected in 5 tertiary hospitals in south China during 2012-2015, and their clinical data were obtained. The clinical characterization was studied by statistical analysis. Whole-genome sequencing and a Galleria mellonella infection model were used to investigate the genetic characterization and pathogenicity of isolates, respectively. Results: Sequence type (ST)457, following ST195, become the second-most prevalent clone in our collection. Patients infected by ST457 had significantly higher 7-day mortality rates (44.4% vs 14.3%; P = .01) and proportions of 7-day deaths (70.6% vs 26.7%; P = .01) than those infected by the other STs of CC92, except for ST195 and ST208. Consistently, the day of death after culture was significantly sooner in patients infected with ST457 than those with the non-ST195/208 members of CC92 (8.71 ± 15.27 vs 25.20 ± 6.51; P = .02). This is accordant with results that ST457 had enhanced virulence with a high mortality rate through use of the G. mellonella larvae infection model. Genomic analysis suggests that ST457 evolved distinctly from the other CC92 members mainly via recombinations. This clone exclusively shared a few virulence factors with the hypervirulence strain LAC-4, including a capsule biosynthesis locus (KL49) that is supposed to be important for the hypervirulence in LAC-4. Conclusions: The rising trends in prevalence and enhanced virulence of ST457 highlight the urgent need for tailored surveillance to control the further dissemination of this clone.

Prevalence of ST1193 clone and IncI1/ST16 plasmid in E-coli isolates carrying blaCTX-M-55 gene from urinary tract infections patients in China.

To study molecular epidemiology of CTX-M-55-carrying Escherichia coli isolates from urinary tract infections (UTIs) in China. 111 blaCTX-M-55-positive E.coli isolates from UTIs patients in China were studied. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homologies among the strains. Conjugation experiments, S1nuclease PFGE and PCR analysis were performed to characterize plasmids harboring blaCTX-M-55 and their genetic environment. 111 isolates were clustered into 86 individual pulsotypes and three clusters by PFGE. Fifty-five (49.5%) of the isolates belonged to 8 STs. Most of the ST1193 isolates belonged to one PFGE cluster. Transconjugants (n = 45) derived from randomly selected blaCTX-M-55 donors (n = 58), were found to contain a single 90-kb conjugative plasmid, which mainly belonged to the IncI1 groups (34, 76%). Among the IncI1 plasmids, the blaCTX-M-55/IncI1/ST16 predominated (23/34, 68%). The blaTEM-1 and aac (3')-II genes were frequently detected on the IncI1 plasmids, and the insertion of ISEcp1 or IS26 was observed at the 48 bp or 45 bp upstream of the start codon of blaCTX-M-55 gene. The dissemination of blaCTX-M-55 gene among E. coli UTI isolates, appeared to be due to both the major clonal lineage of ST1193 and the horizontal transfer of epidemic plasmid IncI1/ST16.

Effects of Fluoroquinolones and Azithromycin on Biofilm Formation of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an opportunistic pathogen that causes respiratory and urinary tract infections, as well as wound infections in immunocompromised patients. This pathogen is difficult to treat due to increased resistance to many antimicrobial agents. We investigated the in vitro biofilm formation of S. maltophilia, including effects of fluoroquinolones (FQs) and azithromycin on biofilm formation. The organism initiated attachment to polystyrene surfaces after a 4 h incubation period, and reached maximal growth at 18-24 h. In the presence of FQs (moxifloxacin, levofloxacin or ciprofloxacin), the biofilm biomass was significantly reduced (P < 0.05). A lower concentration of moxifloxacin (10 µg/mL) exhibited a better inhibiting effect on biofilm formation than 100 µg/mL (P < 0.01), but with no difference in effect compared to the 50 µg/mL concentration (P > 0.05). However, the inhibitory effects of 10 µg/mL of levofloxacin or ciprofloxacin were slightly less pronounced than those of the higher concentrations. A combination of azithromycin and FQs significantly reduced the biofilm inhibiting effect on S. maltophilia preformed biofilms compared to azithromycin or FQs alone. We conclude that early use of clinically acceptable concentrations of FQs, especially moxifloxacin (10 µg/mL), may possibly inhibit biofilm formation by S. maltophilia. Our study provides an experimental basis for a possible optimal treatment strategy for S. maltophilia biofilm-related infections.

Direct detection of methicillin-resistant Staphylococcus aureus in sputum specimens from patients with hospital-associated pneumonia using a novel multilocus PCR assay.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of hospital-associated pneumonia (HAP). The rapid identification of MRSA would be beneficial for early diagnosis. The study aimed to evaluate a multilocus, fluorescence-based PCR assay based on the detection of mecA and nuc genes for identification of S. aureusin lower respiratory tract (LRT) specimens. Sensitivity and specificity of the PCR assay were analyzed. Clinical evaluation for the assay was performed using LRT specimens from patients with HAP, and the sensitivity, specificity, positive and negative predictive values (PPV and NPV) were evaluated in comparison with semi-quantitative culture methods. The result showed the assay provided positive identification of all MRSA reference strains with a limit of detection for MRSA of 4 × 103 CFU/mL. Compared with semi-quantitative culture, the sensitivity, specificity, PPV and NPV were 100%, 89.6%, 75.0%, and 100%, respectively. A positive correlation between MRSA bacterial colonies and PCR copy number was found. The specificity and PPV reached 96.6% and 89.7% respectively, if the PCR copy number reached a definite positive threshold of 5.96 × 105. It suggested that this novel multilocus, fluorescence-based PCR assay proved to be a fast, sensitive and specific tool for direct detection of MRSA from LRT specimens.

The impact of spgM, rpfF, rmlA gene distribution on biofilm formation in Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression. METHODS: The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression. RESULTS: Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains. CONCLUSION: Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.

Glycopeptide minimum inhibitory concentration creep among meticillin-resistant Staphylococcus aureus from 2006-2011 in China.

Vancomycin minimum inhibitory concentration (MIC) creep has recently been demonstrated by many countries but is rarely reported in China. In this study, a total of 1411 meticillin-resistant Staphylococcus aureus (MRSA) isolates were collected from six hospitals in China during the period 2006-2011 and the MICs of vancomycin, teicoplanin and linezolid were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MIC50 and MIC90 values (MICs required to inhibit the growth of 50% and 90% of organisms, respectively) as well as geometric mean (GM) MICs were calculated for all isolates in each year, and MIC creep for the drugs was evaluated. All of the MRSA isolates were susceptible to vancomycin and linezolid. Overall, the vancomycin GM MIC of MRSA isolates was 0.906, 0.952, 0.956, 0.947, 1.013 and 1.040 mg/L, with a significantly increasing trend over the years (P<0.001). Percentages of MRSA isolates with a vancomycin MIC above 1 µg/mL (2 µg/mL≥MIC>1 µg/mL) were 26.0%, 23.5%, 21.6%, 27.8%, 30.6% and 42.8% from 2006-2011, respectively, and increased over time (P<0.005). The teicoplanin GM MIC increased rapidly from 0.749 mg/L in 2008 to 0.973 mg/L in 2011, and ca. 5% of isolates were resistant to teicoplanin in the period according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. MIC shifts were not found for linezolid (P>0.05). In conclusion, a tendency towards decreasing susceptibility to glycopeptides in MRSA has emerged in China.

[Clinical significance of virulence-related genes of type III secretion system of Pseudomonas aeruginosa].

OBJECTIVE: To study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA). METHODS: One hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test. RESULTS: Among the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type. CONCLUSIONS: The TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.

Synthesis and antibacterial activity of C-12 pyrazolinyl spiro ketolides.

A series of C-12 pyrazolinyl spiro ketolide derivatives were designed and synthesized. The C-12 modifications involved replacing the natural C-12 methyl group in clarithromycin core with different pyrazolinyl spiros via chemical synthesis. Potential anti-bacterial activities against both erythromycin-susceptible and erythromycin-resistant bacteria were reported.

[Research of differential expression of virulence-related proteins of T3SS in Pseudomonas aeruginosa].

OBJECTIVE: To analyze the difference of virulence-related protein concerned with type III secretion system (T3SS) in Pseudomonas aeruginosa during the changes of antibiotic sensitivity and interpret the clinical patient data to explore the relationship between the changes in resistance and variance of virulence. METHODS: The isolates of Pseudomonas aeruginosa was isolated from the respiratory tract of a same patient with an altered sensitivity of antibiotics. It turned out to be one clone. The homolog of isolates was determined by ERIC-PCR. The Kirby-Bauer antibiotic testing was employed to detect the sensitivity of antibiotics of isolates. PCR was used to detect the gene of T3SS and virulence of isolates and two-dimensional gel electrophoresis to compare the whole-cell proteins. The mass spectrometry was employed to analyze a variety of protein spots. The relevant information was retrieved from protein databases. Clinical record was collected to study the relationship of clinical features, bacteria resistance and virulence-associated protein. RESULTS: One subject was diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with pneumonia. Pseudomonas aeruginosa was isolated from his respiratory tract three times in one year. Sensitivity spectrum of isolates were as follows: sensitivity, multi-drug resistant (MDR) and pan-drug resistance (PDR). Virulence gene was exo U+/exo S-. Twenty-one differentially expressed proteins were revealed by two-dimensional gel electrophoresis in three isolates. The functions of 11 proteins were definite. Only 9 proteins were associated with basal bacterial metabolism. Disulfide oxidoreductase A (DsbA) corresponded to the variation of virulence and sensitivity spectrum. Clinical record revealed that the severe lung infection was caused by the PDR strain and the patient died within one month. CONCLUSIONS: The sensitivity spectrum and virulence of Pseudomonas aeruginosa may undergo changes when there is an alteration of eco-environment during colonization and infection over a long period of time. DsbA plays a key role in the variety of virulence.

[In vitro antimicrobial activity of piperacillin-sulbactam and other 7 antimicrobial agents against non-fermentative bacilli].

OBJECTIVE: To evaluate the in vitro antimicrobial activity of piperacillin-sulbactam against clinical isolates of non-fermentative bacilli isolated from common infections. METHODS: Microdilution was employed to study the antimicrobial resistance. RESULTS: A total of 770 strains were collected from 6 hospitals in Guangzhou, including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia cepacia, Flavobacterium, and Alcaligenes. Compared with other ß-lactams, piperacillin-sulbactam displayed the highest activity against all the isolates of P.aeruginosa, especially for imipenem non-sensitive isolates, with the susceptibility of 71.9% and 55.8%, respectively. Piperacillin-sulbactam and cefoperazone-sulbactam kept good activity against imipenem sensitive isolates of A.baumannii, with the susceptibility of 71.0% and 73.0%, respectively. For the strains of Burkholderia cepacia, 69% strains exhibited minimal inhibitory concentration (MIC) of ≤ 16 mg/L for piperacillin-sulbactam. For the strains of Flavobacterium, and Alcaligenes, piperacillin-sulbactam also had excellent activity, with the susceptibility of 70.2% and 94.4%, respectively. CONCLUSION: Piperacillin-sulbactam exhibits good activity again non-fermentative bacilli, especially for imipenem non-sensitive isolates of P.aeruginosa.